Production of oil in microorganisms

ABSTRACT

The invention provides methods of cultivating oil-bearing microbes using cellulosic material. Also provided are microorganisms containing one or more exogenous genes that facilitate the use of cellulosic materials as a feedstock. Also provided are microorganisms and methods for manufacturing non-alcohol-based fuels and fuel feedstocks through a process of converting cellulosic materials into oils.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/131,783, filed Jun. 2, 2008, which claims the benefit under 35 U.S.C.119(e) of U.S. Provisional Application No. 60/941,581, filed Jun. 1,2007, U.S. Provisional Application No. 60/959,174, filed Jul. 10, 2007,U.S. Provisional Application No. 60/968,291, filed Aug. 27, 2007, andU.S. Provisional Application No. 61/024,069, filed Jan. 28, 2008, thedisclosures of which are incorporated herein by reference in theirentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing, as filed in U.S.application Ser. No. 12/131,783, in a text file entitledSEQLIS026172002460US.txt, created on Aug. 13, 2008, and containing 27934bytes. The material contained in the text file is hereby incorporated byreference.

FIELD OF THE INVENTION

This disclosure relates to the production of oils, fuels, andoleochemicals made from microorganisms. In particular, the disclosurerelates to oil-bearing microorganisms, including microalgae, yeast andfungi, and to methods of cultivating such microorganisms for theproduction of useful compounds, including lipids, fatty acid esters,fatty acids, aldehydes, alcohols, and alkanes, for use in industry or asan energy or food source. The microorganisms of the invention can beselected or genetically engineered for use in the methods or otheraspects of the invention described herein.

BACKGROUND OF THE INVENTION

Fossil fuel is a general term for buried combustible geologic depositsof organic materials, formed from decayed plants and animals that havebeen converted to crude oil, coal, natural gas, or heavy oils byexposure to heat and pressure in the earth's crust over hundreds ofmillions of years.

In common dialogue, fossil fuel, also known as mineral fuel, is usedsynonymously with other hydrocarbon-containing natural resources such ascoal, oil and natural gas. The utilization of fossil fuels has enabledlarge-scale industrial development and largely supplanted water drivenmills, as well as the combustion of wood or peat for heat. Fossil fuelsare a finite, non-renewable resource.

When generating electricity, energy from the combustion of fossil fuelsis often used to power a turbine. Older generations often used steamgenerated by the burning of the fuel to turn the turbine, but in newerpower plants, the gases produced by burning of the fuel turn a gasturbine directly. With global modernization in the 20th and 21stcenturies, the thirst for energy from fossil fuels, especially gasolinederived from oil, is one of the causes of major regional and globalconflicts.

The burning of fossil fuels by humans is the largest source of emissionsof carbon dioxide, which is one of the greenhouse gases that allowsradiative forcing and contributes to global warming. In the UnitedStates, more than 90% of greenhouse gas emissions come from thecombustion of fossil fuels. In addition, other air pollutants, such asnitrogen oxides, sulfur dioxide, volatile organic compounds (VOCs), andheavy metals are produced.

Human activity raises levels of greenhouse gases primarily by releasingcarbon dioxide from fossil fuel combustion, but other gases, e.g.,methane, are not negligible. The concentrations of several greenhousegases have increased over time due to human activities, such as burningof fossil fuels and deforestation leading to higher carbon dioxideconcentrations. According to the global warming hypothesis, greenhousegases from industry and agriculture have played a major role in therecently observed global warming.

Increased demand for energy by the global economy has also placedincreasing pressure on the cost of hydrocarbons. Aside from energy, manyindustries, including plastics and chemical manufacturers, rely heavilyon the availability of hydrocarbons as a feedstock for theirmanufacturing processes. Cost-effective alternatives to current sourcesof supply could help mitigate the upward pressure on energy and theseraw material costs.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a microbe, which invarious embodiments can comprise a microalgae cell, an oleaginous yeast,or a fungus containing an exogenous gene that encodes a protein selectedfrom the group consisting of a lipase, sucrose transporter, sucroseinvertase, fructokinase, polysaccharide-degrading enzyme, a fattyacyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fattyacyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehydedecarbonylase, and an acyl carrier protein (ACP). The microbe (e.g.,microalgae cell) can, for example, be selected from Table 1. Inparticular embodiments, the cell is a species of the genus Chlorella,such as, e.g., Chlorella fusca, Chlorella protothecoides, Chlorellapyrenoidosa, Chlorella kessleri, Chlorella vulgaris, Chlorellasaccharophila, Chlorella sorokiniana or Chlorella ellipsoidea. In otherembodiments, the microbe is an oleaginous yeast selected from the groupconsisting of Cryptococcus curvatus, Cryptococcus terricolus, Candidasp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis,Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica. Instill other embodiments, the microbe is a fungus selected from the groupconsisting of a species of the genus Mortierella, Mortierrla vinacea,Mortierella alpine, Pythium debaryanum, Mucor circinelloides,Aspergillus ochraceus, Aspergillus terreus, Pennicillium iilacinum, aspecies of the genus Hensenulo, a species of the genus Chaetomium, aspecies of the genus Cladosporium, a species of the genus Malbranchea, aspecies of the genus Rhizopus, and a species of the genus Pythium. Inother embodiments, the invention includes expression of hydrocarbonmodification enzymes in bacterial hosts such as E. Coli and Bacillamethod of producing renewable diesel. In one embodiment, the methodcomprises (a) culturing a population of microorganisms in the presenceof a fixed carbon source, wherein (i) the microorganisms accumulate atleast 10% of their dry cell weight as lipid, and (ii) the fixed carbonsource is selected from the group consisting of glycerol, depolymerizedcellulosic material, sucrose, molasses, glucose, arabinose, galactose,xylose, fructose, arabinose, mannose, acetate, and any combination ofthe foregoing, (b) isolating lipid components from the culturedmicroorganisms, and (c) subjecting the isolated lipid components to oneor more chemical reactions to generate straight chain alkanes, wherebyrenewable diesel is produced.

In another aspect, the present invention is directed to a composition ofliquid hydrocarbons made according to the method described directlyabove, wherein the composition conforms to the specifications of ASTMD975.

In another aspect, the present invention is directed to a method ofproducing jet fuel. In one embodiment, the method comprises (a)culturing a population of microorganisms in the presence of a fixedcarbon source, wherein (i) the microorganisms accumulate at least 10% oftheir dry cell weight as lipid, and (ii) the fixed carbon source isselected from the group consisting of glycerol, depolymerized cellulosicmaterial, sucrose, glucose, arabinose, galactose, xylose, fructose,arabinose, mannose, acetate, and any combination of the foregoing, (b)isolating lipid components from the cultured microorganisms, (c)subjecting the isolated lipid components to one or more chemicalreactions to generate straight chain alkanes, (d) cracking the straightchain alkanes, whereby jet fuel is produced.

In another aspect, the present invention is directed to a composition ofliquid hydrocarbons produced according to the method described directlyabove, wherein the composition conforms to the specifications of ASTMD1655.

In another aspect, the present invention is directed to a microalgae oryeast cell that has been genetically engineered and/or selected toexpress a lipid pathway enzyme at an altered level compared to awild-type cell of the same species. In some cases, the cell producesmore lipid compared to the wild-type cell when both cells are grownunder the same conditions. In some cases, the cell has been geneticallyengineered and/or selected to express a lipid pathway enzyme at a higherlevel than the wild-type cell. In some cases, the lipid pathway enzymeis selected from the group consisting of pyruvate dehydrogenase,acetyl-CoA carboxylase, acyl carrier protein, and glycerol-3 phosphateacyltransferase. In some cases, the cell has been genetically engineeredand/or selected to express a lipid pathway enzyme at a lower level thanthe wild-type cell. In at least one embodiment in which the cellexpresses the lipid pathway enzyme at a lower level, the lipid pathwayenzyme comprises citrate synthase.

In some embodiments, the microalgae or yeast cell described above hasbeen genetically engineered and/or selected to express a globalregulator of fatty acid synthesis at an altered level compared to thewild-type cell, whereby the expression levels of a plurality of fattyacid synthetic genes are altered compared to the wild-type cell. In somecases, the lipid pathway enzyme comprises an enzyme that modifies afatty acid. In some cases, the lipid pathway enzyme is selected from astearoyl-ACP desaturase and a glycerolipid desaturase.

In another aspect, the present invention is directed to an oil-producingmicrobe containing one or more exogenous genes, wherein the exogenousgenes encode protein(s) selected from the group consisting of a fattyacyl-ACP thioesterase, a fatty acyl-CoA reductase, a fatty aldehydereductase, a fatty acyl-CoA/aldehyde reductase, a fatty aldehydedecarbonylase, and an acyl carrier protein. In some cases, the microbeis Chlorella protothecoides, Chlorella minutissima, Chlorella emersonii,Chlorella sorokiniana, Chlorella ellipsoidea, or Chlorella sp. In othercases, the microbe is another species as described herein. In oneembodiment, the exogenous gene is in operable linkage with a promoter,which is inducible or repressible in response to a stimulus. In somecases, the stimulus is selected from the group consisting of anexogenously provided small molecule, heat, cold, and light. In somecases, the exogenous gene is expressed in a cellular compartment. Insome embodiments, the cellular compartment is selected from the groupconsisting of a chloroplast and a mitochondrion.

In one embodiment, the exogenous gene encodes a fatty acid acyl-ACPthioesterase. In some cases, the thioesterase encoded by the exogenousgene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an acylcarrier protein (ACP). In some cases, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acidfrom an ACP. In one embodiment, the thioesterase encoded by theexogenous gene catalyzes the cleavage of a 12-carbon fatty acid from anACP.

In one embodiment, the exogenous gene encodes a fatty acyl-CoA/aldehydereductase. In some cases, the reductase encoded by the exogenous genecatalyzes the reduction of a 20 to 30-carbon fatty acyl-CoA to acorresponding primary alcohol. In some cases, the reductase encoded bythe exogenous gene catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding primary alcohol. In some cases, thereductase encoded by the exogenous gene catalyzes the reduction of a 10to 14-carbon fatty acyl-CoA to a corresponding primary alcohol. In oneembodiment, the reductase encoded by the exogenous gene catalyzes thereduction of a 12-carbon fatty acyl-CoA to dodecanol.

In one embodiment, the exogenous gene encodes a fatty acyl-CoAreductase. In some cases, the reductase encoded by the exogenous genecatalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to acorresponding aldehyde. In one embodiment, the reductase encoded by theexogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA tododecanal.

In at least one embodiment, the microbe of the invention furthercontains one or more exogenous sucrose utilization genes.

In another aspect, the present invention is directed to a microbecontaining two exogenous genes, wherein a first exogenous gene encodes afatty acyl-ACP thioesterase and a second exogenous gene encodes aprotein selected from the group consisting of a fatty acyl-CoAreductase, a fatty acyl-CoA/aldehyde reductase, and an acyl carrierprotein. In some cases, the microbe is Chlorella minutissima, Chlorellaemersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp.,or Chlorella protothecoides. In other cases, the microbe is anotherspecies as described herein. In some cases, the two exogenous genes areeach in operable linkage with a promoter, which is inducible in responseto a stimulus. In some cases, each promoter is inducible in response toan identical stimulus.

In one embodiment, the thioesterase encoded by the first exogenous genecatalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP. Insome embodiments, the second exogenous gene encodes a fattyacyl-CoA/aldehyde reductase which catalyzes the reduction of an 8 to18-carbon fatty acyl-CoA to a corresponding primary alcohol. In somecases, the thioesterase encoded by the first exogenous gene catalyzesthe cleavage of a 10 to 14-carbon fatty acid from an ACP, and thereductase encoded by the second exogenous gene catalyzes the reductionof a 10 to 14-carbon fatty acyl-CoA to the corresponding primaryalcohol, wherein the thioesterase and the reductase act on the samecarbon chain length. In one embodiment, the thioesterase encoded by thefirst exogenous gene catalyzes the cleavage of a 12-carbon fatty acidfrom an ACP, and the reductase encoded by the second exogenous genecatalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol. Insome embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase which catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding aldehyde.

In some embodiments, the second exogenous gene encodes a fatty acyl-CoAreductase, and the microbe further contains a third exogenous geneencoding a fatty aldehyde decarbonylase. In some cases, the thioesteraseencoded by the first exogenous gene catalyzes the cleavage of an 8 to18-carbon fatty acid from an ACP, the reductase encoded by the secondexogenous gene catalyzes the reduction of an 8 to 18-carbon fattyacyl-CoA to a corresponding fatty aldehyde, and the decarbonylaseencoded by the third exogenous gene catalyzes the conversion of an 8 to18-carbon fatty aldehyde to a corresponding alkane, wherein thethioesterase, the reductase, and the decarbonylase act on the samecarbon chain length.

In some embodiments, the second exogenous gene encodes an acyl carrierprotein that is naturally co-expressed with the fatty acyl-ACPthioesterase.

In some embodiments, the second exogenous gene encodes an acyl carrierprotein, and the microbe further contains a third exogenous geneencoding a protein selected from the group consisting of a fattyacyl-CoA reductase and a fatty acyl-CoA/aldehyde reductase. In somecases, the third exogenous gene encodes a fatty acyl-CoA reductase, andthe microbe further contains a fourth exogenous gene encoding a fattyaldehyde decarbonylase.

In another aspect, the present invention is directed to a method ofproducing a molecule in a microbe population. In one embodiment, themethod comprises culturing a population of microbes in a culture medium,wherein the microbes contain (i) a first exogenous gene encoding a fattyacyl-ACP thioesterase, and (ii) a second exogenous gene encoding a fattyacyl-CoA/aldehyde reductase, and the microbes synthesize a fatty acidlinked to an acyl carrier protein (ACP), the fatty acyl-ACP thioesterasecatalyzes the cleavage of the fatty acid from the ACP to yield, throughfurther processing, a fatty acyl-CoA, and the fatty acyl-CoA/aldehydereductase catalyzes the reduction of the acyl-CoA to an alcohol.

In one embodiment of the method of producing a molecule in a microbepopulation, the microbe is Chlorella minutissima, Chlorella emersonii,Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. or Chlorellaprotothecoides. In other cases, the microbe is another species ofmicroorganism as described herein. In some cases, the culture mediumcontains glycerol. In one embodiment, the glycerol is a byproduct of atransesterification process. In some cases, the culture medium containsglycerol and at least one other fixed carbon source. In one embodiment,the at least one other fixed carbon source is sucrose. In some cases,all of the glycerol and all of the at least one other fixed carbonsource are provided to the microbes at the beginning of fermentation. Insome cases, the glycerol and the at least one other fixed carbon sourceare fed to the microbes at a predetermined rate over the course offermentation. In some culture methods of the invention, glycerol isprovided to the microbes in the absence of the at least one other fixedcarbon source for a first period of time, the at least one other fixedcarbon source is provided at the end of the first period of time, andthe microbes are cultured for a second period of time in the presence ofthe at least one other fixed carbon source.

In some embodiments, the exogenous genes are in operable linkage with apromoter that is inducible in response to a first stimulus. In somecases, the method further comprises providing the first stimulus, andincubating the population of microbes for a first period of time in thepresence of the first stimulus to produce an alcohol. In some cases, themethod further comprises extracting the alcohol from aqueous biomasscomprising the culture medium and the microbes.

In some embodiments, the thioesterase encoded by the first exogenousgene catalyzes the cleavage of an 8 to 18-carbon fatty acid from theACP, and the reductase encoded by the second exogenous gene catalyzesthe reduction of an 8 to 18-carbon fatty acyl-CoA to a correspondingprimary alcohol, wherein the thioesterase and the reductase act on thesame carbon chain length. In some cases, the thioesterase encoded by thefirst exogenous gene catalyzes the cleavage of an 10 to 14-carbon fattyacid from the ACP, and the reductase encoded by the second exogenousgene catalyzes the reduction of an 10 to 14-carbon fatty acyl-CoA to acorresponding primary alcohol, wherein the thioesterase and thereductase act on the same carbon chain length. In one embodiment, thethioesterase encoded by the first exogenous gene catalyzes the cleavageof a 12-carbon fatty acid from the ACP, and the reductase encoded by thesecond exogenous gene catalyzes the reduction of a 12-carbon fattyacyl-CoA to dodecanol. In some cases, the microbes further contain athird exogenous gene encoding an acyl carrier protein. In someembodiments, the third exogenous gene encodes an acyl carrier proteinthat is naturally co-expressed with the fatty acyl-ACP thioesterase.

In another aspect, the present invention is directed to a method ofproducing a lipid molecule in a microbe population. In one embodiment,the method comprises culturing a population of microbes in a culturemedium, wherein the microbes contain (i) a first exogenous gene encodinga fatty acyl-ACP thioesterase, and (ii) a second exogenous gene encodinga fatty acyl-CoA reductase, and wherein the microbes synthesize a fattyacid linked to an acyl carrier protein (ACP), the fatty acyl-ACPthioesterase catalyzes the cleavage of the fatty acid from the ACP toyield, through further processing, a fatty acyl-CoA, and the fattyacyl-CoA reductase catalyzes the reduction of the acyl-CoA to analdehyde. In some cases, the microbe is Chlorella minutissima, Chlorellaemersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp.,or Chlorella protothecoides. In other cases, the microbe is anotherspecies of microorganism as described herein.

In some embodiments, the exogenous genes are in operable linkage with apromoter that is inducible in response to a first stimulus, and themethod further comprises providing the first stimulus, and incubatingthe population of microbes for a first period of time in the presence ofthe first stimulus to produce an aldehyde. In one embodiment, the methodfurther comprises extracting the aldehyde from aqueous biomasscomprising the culture medium and the population of microbes.

In some embodiments, the thioesterase encoded by the first exogenousgene catalyzes the cleavage of an 8 to 18-carbon fatty acid from theACP, and the reductase encoded by the second exogenous gene catalyzesthe reduction of an 8 to 18-carbon fatty acyl-CoA to a correspondingaldehyde, wherein the thioesterase and the reductase act on the samecarbon chain length. In some cases, the microbes further contain a thirdexogenous gene encoding a fatty aldehyde decarbonylase that catalyzesthe conversion of the aldehyde to an alkane.

In some cases, the exogenous genes are in operable linkage with apromoter that is inducible in response to a first stimulus, and themethod further comprises providing the first stimulus, and incubatingthe population of microbes for a first period of time in the presence ofthe first stimulus to produce an alkane. In some cases, the methodfurther comprises extracting the alkane from aqueous biomass comprisingculture medium and the microbe population.

In some cases, the thioesterase encoded by the first exogenous genecatalyzes the cleavage of an 8 to 18-carbon fatty acid from the ACP, thereductase encoded by the second exogenous gene catalyzes the reductionof an 8 to 18-carbon fatty acyl-CoA to a corresponding aldehyde, and thedecarbonylase encoded by the third exogenous gene catalyzes theconversion of an 8 to 18-carbon aldehyde to a corresponding alkane,wherein the thioesterase, the reductase, and the decarbonylase act onthe same carbon chain length. In some embodiments, the microbes furthercontain a third exogenous gene encoding an acyl carrier protein. In somecases, the third exogenous gene encodes an acyl carrier protein that isnaturally co-expressed with the fatty acyl-ACP thioesterase. In somecases, the microbes further contain a fourth exogenous gene encoding afatty aldehyde decarbonylase that catalyzes the conversion of thealdehyde to an alkane.

In some methods, the culture medium contains glycerol. In oneembodiment, the glycerol is a byproduct of a transesterificationprocess. In some cases, the culture medium contains glycerol and atleast one other fixed carbon source. In one embodiment, the at least oneother fixed carbon source is sucrose. In some cases, all of the glyceroland all of the at least one other fixed carbon source are provided tothe microbes at the beginning of fermentation. In some cases, theglycerol and the at least one other fixed carbon source are fed to themicrobes at a predetermined rate over the course of fermentation. In oneembodiment, glycerol is provided to the microbes in the absence of theat least one other fixed carbon source for a first period of time, theat least one other fixed carbon source is provided at the end of thefirst period of time, and the microbes are cultured for a second periodof time in the presence of the at least one other fixed carbon source.

In another aspect, the present invention is directed to a method ofproducing a fatty acid molecule having a specified carbon chain lengthin a microbe population. In one embodiment, the method comprisesculturing a population of lipid-producing microbes in a culture medium,wherein the microbes contain an exogenous gene encoding a fatty acyl-ACPthioesterase having an activity specific to a carbon chain length, andwherein the microbes synthesize a fatty acid linked to an acyl carrierprotein (ACP) and the thioesterase catalyzes the cleavage of the fattyacid from the ACP when the fatty acid has been synthesized to thespecific carbon chain length. In some cases, the microbe is Chlorellaminutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorellaellipsoidea, Chlorella sp., or Chlorella protothecoides. In other cases,the microbe is another species of microorganism as described herein.

In some embodiments, the exogenous gene is in operable linkage with apromoter that is inducible in response to a first stimulus, and themethod further comprises providing the first stimulus, and incubatingthe population of microbes for a period of time in the presence of thefirst stimulus. In some cases, the method further comprises extractingthe fatty acid from aqueous biomass comprising culture medium and themicrobe population.

In some cases, the microbes further contain a second exogenous geneencoding an acyl carrier protein. In some embodiments, the secondexogenous gene encodes an acyl carrier protein that is naturallyco-expressed with the fatty acyl-ACP thioesterase. In one embodiment,the acyl-ACP thioesterase catalyzes the cleavage of an 8 to 18-carbonfatty acid from the ACP.

In some cases, the culture medium contains glycerol. In one embodiment,the glycerol is a byproduct of a transesterification process. In someembodiments, the culture medium contains glycerol and at least one otherfixed carbon source. In one embodiment, the at least one other carbonsource is sucrose. In some cases, all of the glycerol and all of the atleast one other fixed carbon source are provided to the microbes at thebeginning of fermentation. In some cases, the glycerol and the at leastone other fixed carbon source are fed to the microbes at a predeterminedrate over the course of fermentation. In one embodiment, glycerol isprovided to the microbes in the absence of the at least one other fixedcarbon source for a first period of time, the at least one other fixedcarbon source is provided at the end of the first period of time, andthe microbes are cultured for a second period of time in the presence ofthe at least one other fixed carbon source.

In another aspect, the present invention is directed to a microalgaecell containing an exogenous gene, wherein the exogenous gene encodes aprotein selected from the group consisting of a lipase, a sucrosetransporter, a sucrose invertase, a fructokinase, or apolysaccharide-degrading enzyme. In some cases, the cell is Chlorellaminutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorellaellipsoidea, Chlorella sp., or Chlorella protothecoides. In other cases,the cell is another species of microalgae as described herein.

In some cases, the exogenous gene is in operable linkage with apromoter. In some cases, the promoter is inducible or repressible inresponse to a stimulus. In various embodiments, the stimulus is selectedfrom the group consisting of an exogenously provided small molecule,heat, cold, and light. In some cases, the exogenous gene is expressed ina cellular compartment. In some embodiments, the cellular compartment isselected from the group consisting of a chloroplast and a mitochondrion.

In some cases, the gene encodes a lipase that has at least 70% aminoacid identity with a lipase selected from Table 9. In one embodiment,the lipase is novozym-435. In one embodiment, thepolysaccharide-degrading enzyme is endogenous to a Chlorella virus.

In another aspect, the present invention is directed to a microalgaecell containing two exogenous genes, wherein a first exogenous geneencodes a lipase and a second exogenous gene encodes apolysaccharide-degrading enzyme. In some cases, the exogenous genes areeach in operable linkage with a promoter. In some cases, the exogenousgenes are each in operable linkage with promoters that are inducible inresponse to a stimulus. In some cases, the exogenous genes are each inoperable linkage with promoters that are inducible in response to thesame stimulus. In some cases, the exogenous genes are each in operablelinkage with a promoter that is inducible in response to at least onestimulus that does not induce the other promoter.

In another aspect, the present invention is directed to a method ofmanufacturing a lipid molecule in a microbe. In one embodiment, themethod comprises (a) culturing the microbe for a first period of timesufficient to increase the cell density, wherein the microbe contains(i) an exogenous gene encoding a lipase, and/or (ii) an exogenous geneencoding a polysaccharide-degrading enzyme, wherein the exogenousgene(s) are in operable linkage with a promoter that is inducible inresponse to a stimulus, (b) providing the stimulus, and (c) incubatingthe microbe for a second period of time in the presence of the stimulus.

In another aspect, the present invention is directed to a method ofmanufacturing a lipid molecule in a microbe. In one embodiment, themethod comprises (a) culturing a lipid-producing microbe for a firstperiod of time sufficient to increase the cell density, (b) providing avirus capable of infecting and lysing the microbe when in direct contactwith the microbe, and (c) incubating the microbe for a second period oftime to produce lysed aqueous biomass. In one embodiment, the methodfurther comprises extracting lipid molecules from the lysed aqueousbiomass.

In another aspect, the present invention is directed to a microalgaecell containing an exogenous gene, wherein the exogenous gene encodes acofactor for a lipid pathway enzyme or encodes a protein thatparticipates in the synthesis of the cofactor.

In another aspect, the present invention is directed to a method ofculturing a lipid-producing microbe. In one embodiment, the methodcomprises culturing the microbe in the presence of a sufficient amountof one or more cofactor(s) for a lipid pathway enzyme to increasemicrobial lipid yield over microbial lipid yield in the absence of saidone or more cofactors. In some cases, the one or more cofactors is avitamin required by one or more lipid pathway enzymes. In oneembodiment, the one or more cofactors is biotin. In some cases, the oneor more cofactors is/are provided by including in the culture a microbethat has been genetically engineered to produce the one or morecofactors.

In another aspect, the present invention is directed to a method offermenting a microorganism, which comprises providing a mixturecomprising glucose and xylose as an energy source to the microorganism.In one embodiment, the mixture further comprises lignin. In oneembodiment, the mixture further comprises at least one species offurfural. In some cases, the mixture is depolymerized cellulosicmaterial. In some cases, the mixture further comprises as least onesucrose utilization enzyme. In one embodiment, the mixture comprises asucrose invertase.

In some cases, the microorganism is selected from the group consistingof Bracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri,Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorellaminutissima, Chlorella ovalis, Chlorella protothecoides, Chlorellasaccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris,Parachlorella kessleri, Prototheca moriformis, and Pseudochlorellaaquatica. In other cases, the microorganism is another species ofmicroorganism as described herein. In some cases, the microorganism hasbeen genetically engineered to express an exogenous gene encoding atleast one lipid modification enzyme, hydrocarbon modification enzyme, orsucrose utilization enzyme.

In another aspect, the present invention is directed to a method ofculturing a microalgae, which comprises culturing the microalgae in aculture medium including a feedstock comprising at least one carbonsubstrate selected from the group consisting of a cellulosic material, a5-carbon sugar, a 6-carbon sugar, and acetate. In some cases, the carbonsubstrate is glucose and the microalgae is of a genus selected from thegroup consisting of Chlorella, Parachlorella, Pseudochlorella,Bracteococcus, Prototheca and Scenedesmus. In some cases, the carbonsubstrate is xylose and the microalgae is of a genus selected from thegroup consisting of Chlorella, Pseudochlorella, and Prototheca. In somecases, the carbon substrate is sucrose and the microalgae is of a genusselected from the group consisting of Chlorella, and Bracteococcus. Insome cases, the carbon substrate is fructose and the microalgae is of agenus selected from the group consisting of Chlorella, Parachlorella,Prototheca, and Scenedesmus. In some cases, the carbon substrate isarabinose and the microalgae is Chlorella sp. In some cases, the carbonsubstrate is mannose and the microalgae is of a genus selected from thegroup consisting of Chlorella, Parachlorella, Bracteococcus, Prototheca,and Scenedesmus. In some cases, the carbon substrate is galactose andthe microalgae is of a genus selected from the group consisting ofBracteococcus, Parachlorella, Chlorella, Pseudochlorella, Bracteococcus,and Prototheca. In some cases, the carbon substrate is acetate and themicroalgae is of a genus selected from the group consisting ofChlorella, Parachlorella, and Prototheca.

In one embodiment, the culture medium further includes at least onesucrose utilization enzyme. In some cases, the microalgae has beengenetically engineered to express an exogenous gene encoding at leastone lipid modification enzyme, hydrocarbon modification enzyme, orsucrose utilization enzyme. In some cases, the culture medium includes asucrose invertase.

In another aspect, the present invention is directed to a method ofculturing microalgae comprising placing a plurality of microalgae cellsin the presence of depolymerized cellulosic material. In some cases, themicroalgae are cultured in the presence of an additional fixed carbonsource selected from the group consisting of glycerol, sucrose, glucose,arabinose, galactose, xylose, fructose, arabinose, mannose, acetate, andany combination of the foregoing. In one embodiment, the microalgae arecultured in the presence of at least one sucrose utilization enzyme.

In some cases, the microalgae is selected from a species of the genusBracteococcus, a species of the genus Chlorella, a species of the genusParachlorella, a species of the genus Prototheca, or a species of thegenus Pseudochlorella. In some cases, the microalgae is selected fromBracteococcus minor, Chlorella ellipsoidea, Chlorella kessleri,Chlorella luteoviridis, Bracteococcus medionucleatus, Chlorellaminutissima, Chlorella ovalis, Chlorella protothecoides, Chlorellasaccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris,Parachlorella kessleri, Prototheca moriformis, and Pseudochlorellaaquatica. In other cases, the microalgae is another species ofmicroalgae as described herein.

In some embodiments, the microalgae has been genetically engineered toexpress an exogenous gene encoding at least one lipid modificationenzyme, hydrocarbon modification enzyme, or sucrose utilization enzyme.In one embodiment, the at least one sucrose utilization enzyme is asucrose invertase. In some cases, the at least one lipid modificationenzyme is selected from a stearoyl-ACP desaturase, a glycerolipiddesaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, and aglycerol-3 phosphate acyltransferase. In some cases, the at least onehydrocarbon modification enzyme is selected from a fatty acyl-ACPthioesterase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, afatty acyl-CoA/aldehyde reductase, a fatty aldehyde decarbonylase, andan acyl carrier protein.

In another aspect, the present invention is directed to a method ofculturing a lipid-producing microbe, the method comprising culturing themicrobe in the presence of acetic acid and the absence of a fixednitrogen source. In some cases, the microbe is cultured in the presenceof a sufficient amount of acetic acid to increase microbial lipid yieldover microbial lipid yield in the absence of acetic acid, whereinculture conditions are otherwise the same between the two cultures.

In another aspect, the present invention is directed to a microbialculture containing a population of microorganisms and a culture mediumcomprising glucose, xylose, and a molecule selected from the groupconsisting of lignin and a species of furfural. In some cases, themicroorganisms are selected from Bracteococcus minor, Chlorellaellipsoidea, Chlorella kessleri, Chlorella luteoviridis, Bracteococcusmedionucleatus, Chlorella minutissima, Chlorella ovalis, Chlorellaprotothecoides, Chlorella saccharophila, Chlorella sorokiniana,Chlorella sp., Chlorella vulgaris, Parachlorella kessleri, Protothecamoriformis, and Pseudochlorella aquatica. In other cases, themicroorganisms are another species of microorganism as described herein.

In another aspect, the present invention is directed to a method ofcultivating microalgae. In one embodiment, the method comprises (a)providing a microalgae cell capable of performing heterotrophic growth,(b) placing the microalgae cell in growth media, wherein the growthmedia comprises depolymerized cellulosic material, and (c) incubatingthe microalgae for a period of time sufficient to allow the cell togrow.

In another aspect, the present invention is directed to a method ofbiodiesel manufacturing. In one embodiment, the method comprises (a)culturing a lipid-producing microorganism in a first microbial culture,(b) recovering lipid from the biomass produced by the first microbialculture, (c) subjecting the lipid to transesterification to producefatty acid ester(s) and glycerol, and (d) adding the glycerol to asecond microbial culture. In some cases, the first and second microbialcultures are cultures of the same species of microorganism. In somecases, the second microbial culture comprises microorganisms selectedfrom the group consisting of Parachlorella kessleri, Chlorellaprotothecoides, Bracteococcus medionucleatus, Prototheca moriformis,Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana. Inother cases, the second microbial culture comprises another species ofmicroorganism as described herein.

In another aspect, the present invention is directed to a method offermentation comprising culturing a microorganism in the presence ofglycerol and at least one other fixed carbon source. In some cases, theglycerol and the at least one other fixed carbon source are provided tothe microorganism simultaneously at a predetermined ratio. In somecases, all of the glycerol and the at least one other fixed carbonsource are provided to the microorganism at the beginning of thefermentation. In some cases, all of the glycerol and the at least oneother fixed carbon source are fed to the microorganism at apredetermined rate over the course of the fermentation. In oneembodiment of the method, glycerol is provided to the microorganism inthe absence of the at least one other fixed carbon source for a firstperiod of time, the at least one other fixed carbon source is providedat the end of the first period of time, and the microorganism iscultured for a second period of time in the presence of the at least oneother fixed carbon source. In one embodiment, the at least one otherfixed carbon source is fed to the microorganism at a predetermined rateduring the second period of time. In some cases, all of the at least oneother fixed carbon source is provided to the microorganism at the end ofthe first period of time. In one embodiment of the method, the at leastone other fixed carbon source is provided to the microorganism in theabsence of glycerol for a first period of time, glycerol is provided atthe end of the first period of time, and the microorganism is culturedfor a second period of time in the presence of glycerol. In oneembodiment, the glycerol is a byproduct of a transesterificationprocess. In one embodiment, the glycerol is acidulated. In anotherembodiment, the glycerol is non-acidulated. In some cases, the at leastone other fixed carbon source is glucose. In some cases, the at leastone other fixed carbon source is depolymerized cellulosic material. Inone embodiment, the at least one other fixed carbon source is sucrose.

In another aspect, the present invention is directed to a fermentorcomprising a population of microorganisms, glycerol, and at least onesugar selected from the group consisting of xylose, glucose, andsucrose. In one embodiment, the glycerol is a byproduct of a lipidtransesterification process. In some cases, the microorganisms areselected from the group consisting of Parachlorella kessleri, Chlorellaprotothecoides, Bracteococcus medionucleatus, Prototheca moriformis,Chlorella minutissima, Chlorella sp., and Chlorella sorokiniana. Inother cases, the microorganisms are another species as described herein.

In another aspect, the present invention is directed to a method offermenting a microorganism. In one embodiment, the method comprisesproviding byproduct glycerol from a transesterification process as asole source of fixed carbon energy. In one embodiment, no light energyis provided to the microorganism. In another embodiment, light energy isprovided to the microorganism. In some cases, the microorganism isselected from Parachlorella kessleri, Chlorella protothecoides,Bracteococcus medionucleatus, Prototheca moriformis, Chlorellaminutissima, Chlorella sp., and Chlorella sorokiniana. In other cases,the microorganism is another species as described herein.

In another aspect, the present invention is directed to a microorganismcontaining an exogenous sucrose utilization gene. In one embodiment, thegene encodes a sucrose transporter. In one embodiment, the gene encodesa sucrose invertase. In one embodiment, the gene encodes a fructokinase.In some cases, the microorganism is a species selected from the groupconsisting of Chlorella minutissima, Chlorella emersonii, Chlorellasorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorellaprotothecoides. In other cases, the microorganism is another species asdescribed herein.

In another aspect, the present invention is directed to a cell of thespecies Chlorella protothecoides, Chlorella emersonii, or Chlorellaminutissima wherein the cell contains an exogenous gene. In some cases,the exogenous gene encodes a protein selected from the group consistingof a sucrose transporter, a sucrose invertase, a lipid modificationenzyme, a hydrocarbon modification enzyme and a fructokinase. In someembodiments, the protein is a sucrose invertase secreted into theextracellular space. In some embodiments, the protein is a sucroseinvertase targeted to the cytoplasm.

In another aspect, the present invention is directed to a microbialculture containing a population of microorganisms, and a culture mediumcomprising (i) sucrose and (ii) a sucrose invertase enzyme.

In another aspect, the present invention is directed to a microbialculture containing a population of microorganisms, and a culture mediumcomprising (i) molasses and (ii) a sucrose invertase enzyme.

In another aspect, the present invention is directed to a microbialculture containing a population of microorganisms, and a culture mediumcomprising (i) sucrose, (ii) lignin, and (iii) a sucrose invertaseenzyme.

In the various microbial cultures described above, the microorganismscontain at least one exogenous sucrose utilization gene. In someembodiments, the sucrose utilization gene encodes a sucrose transporter,a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase. Inone embodiment, the sucrose invertase enzyme is a secrectable sucroseinvertase enzyme encoded by an exogenous sucrose invertase geneexpressed by the population of microorganisms. In some cases, themicroorganisms contain at least one exogenous gene encoding a lipidpathway enzyme or a hydrocarbon modification enzyme.

In another aspect, the present invention is directed to a nucleic acidcomprising a cDNA encoding a sucrose utilization gene, and a cDNAencoding a protein conferring resistance to the antibiotic hygromycin orthe antibiotic G418.

In embodiments of the various methods, compositions, cells,microorganisms, microbes, microbial cultures, fermentors, and the like,described above, the microorganism or microbe can be a microalgae, anoleaginous yeast, a fungus, or a bacterium, unless otherwise specified.In some cases, the microorganism is selected from the group consistingof the microalgae listed in Table 1. In some cases, the microorganism isa species of the genus Chlorella. In some cases, the microorganism isselected from the group consisting of Chlorella anitrata, Chlorellaantarctica, Chlorella aureoviridis, Chlorella candida, Chlorellacapsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorellaemersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorellaglucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila,Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorellaphotophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorellaregularis, Chlorella regularis var. minima, Chlorella regularis var.umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorellasaccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex,Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorellavulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis,Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f.tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorellaxanthella, and Chlorella zofingiensis. In some cases, the microorganismis an oleaginous yeast selected from the group consisting ofCryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomycesstarkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorulaglutinis, Rhodotorula gracilis, and Yarrowia lipolytica. In some cases,the microorganism is a fungus selected from the group consisting of aspecies of the genus Mortierella, Mortierrla vinacea, Mortierellaalpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus,Aspergillus terreus, Pennicillium iilacinum, a species of the genusHensenulo, a species of the genus Chaetomium, a species of the genusCladosporium, a species of the genus Malbranchea, a species of the genusRhizopus, and a species of the genus Pythium.

In the various embodiments described above, the microorganism cancontain at least one exogenous sucrose utilization gene. In some cases,the sucrose utilization gene encodes a sucrose transporter, a sucroseinvertase, a hexokinase, a glucokinase, or a fructokinase.

In the various embodiments described above, the microorganism cancontain at least one exogenous gene encoding a lipid pathway enzyme. Insome cases, the lipid pathway enzyme is selected from the groupconsisting of a stearoyl-ACP desaturase, a glycerolipid desaturase, apyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrierprotein, and a glycerol-3 phosphate acyltransferase.

In the various embodiments described above, the microorganism cancontain at least one exogenous gene encoding a hydrocarbon modificationenzyme. In some cases, the hydrocarbon modification enzyme is selectedfrom the group consisting of a fatty acyl-ACP thioesterase, a fattyacyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fattyaldehyde reductase, a fatty aldehyde decarbonylase, and/or an acylcarrier protein.

Any two or more of the various embodiments described above can becombined together to produce additional embodiments encompassed withinthe scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dry cell weight per liter of multiple species and strainsof Chlorella when cultured in the presence of various types of glycerolwith and without additional glucose.

FIG. 2 shows dry cell weight per liter of multiple species and strainsof Chlorella when cultured in the presence of various types of glycerolwith additional glucose.

FIG. 3 shows relative lipid concentration of cultures of multiplespecies and strains of Chlorella when cultured in the presence ofvarious types of glycerol with additional glucose.

FIG. 4 shows lipid concentration of cultures of multiple species andstrains of Chlorella when cultured in the presence of various types ofglycerol with and without additional glucose.

FIG. 5 shows lipid as a percent of dry cell weight of two species andstrains of Chlorella when cultured in the presence of various types ofglycerol with additional glucose, wherein glycerol is added sequentiallyafter glucose.

FIG. 6 shows lipid as a percent of dry cell weight of two species andstrains of Chlorella when cultured in the presence of various types ofglycerol with additional glucose.

FIG. 7 shows relative lipid concentration of cultures of multiplespecies and strains of Chlorella when cultured in the presence of 2%glucose and 1% glucose+1% reagent grade glycerol.

FIG. 8 shows lipid as a percent of dry cell weight of multiple speciesand strains of Chlorella when cultured in the presence of glucose withand without reagent grade glycerol, wherein glycerol is addedsequentially or in combination with glucose.

FIG. 9 shows relative lipid concentration of cultures of multiplespecies and strains of Chlorella when cultured in the presence ofvarious types of glycerol with additional glucose, wherein glycerol isadded sequentially or in combination with glucose.

FIG. 10 shows dry cell weight per liter of multiple species and strainsof Chlorella when cultured in the presence of various types of glycerolwith additional glucose, wherein glycerol is added sequentially or incombination with glucose.

FIG. 11( a) shows lipid as a percent of dry cell weight of Spirulinaplatensis when cultured in the presence of glucose, reagent gradeglycerol, non-acidulated biodiesel byproduct glycerol, and a combinationof glycerol and glucose.

FIG. 11( b) shows lipid as a percent of dry cell weight of Naviculapelliculosa when cultured in the presence of various types of glyceroland in the presence of combinations of glycerol and glucose.

FIG. 12( a) shows lipid as a percent of dry cell weight of Scenedesmusarmatus when cultured in the presence of various types of glycerol andin the presence of a combination of glycerol and glucose.

FIG. 12( b) shows dry cell weight per liter of Scenedesmus armatus whencultured in the presence of various types of glycerol and in thepresence of a combination of biodiesel byproduct glycerol and glucose.

FIG. 13 shows dry cell weight per liter of Navicula pelliculosa whencultured in the presence of various types of glycerol and in thepresence of a combination of non-acidulated biodiesel byproduct glyceroland glucose.

FIG. 14 shows dry cell weight per liter of Scenedesmus armatus andNavicula pelliculosa when cultured in the presence of acidulated andnon-acidulated biodiesel byproduct glycerol with additional glucose,wherein glycerol is added sequentially or in combination with glucose.

FIG. 15 shows a synergistic effect of a combination of xylose andglucose on growth of Chlorella compared to xylose or glucose alone.

FIG. 16 shows genotyping of Chlorella protothecoides transformantscontaining an exogenous gene.

FIG. 17 shows codon usage of Chlorella protothecoides.

FIG. 18 shows codon usage of D. salina and Chlorella pyrenoidosa.

FIG. 19 shows (a) reagent grade glycerol; (b) non-acidulated biodieselbyproduct glycerol; and (c) acidulated biodiesel byproduct glycerol, allof which were used in experiments described in the Examples.

FIG. 20 shows growth of Chlorella protothecoides on glucose andfructose.

FIG. 21 shows growth of Chlorella fusca on 1% sucrose.

FIG. 22 shows growth of Chlorella kessleri on 1% sucrose.

FIG. 23 shows dry cell weight per liter of Chlorella protothecoides whencultured in the presence of glucose, sucrose, or one of several molassessamples (designated BS1, BS2 and HTM) in the presence or absence of asucrose invertase.

FIG. 24 shows growth of Chlorella protothecoides when cultured in thepresence of glucose, sucrose, or one of several molasses samples(designated BS1, BS2 and HTM) in the presence or absence of a sucroseinvertase as measured by relative cell density.

FIG. 25 shows an illustration of various plasmid constructs of yeastinvertase (SUC2) with three different promoters (designated CMV, CV andHUP1) as well as restriction sites useful for subcloning.

FIG. 26 shows genotyping of Chlorella protothecoides transformantsselected on sucrose in the dark containing an exogenous sucroseinvertase gene.

FIG. 27 shows genotyping of Chlorella protothecoides cells transformedwith a gene encoding a secreted sucrose invertase from S. cerevisiae.

FIG. 28 shows genotyping of Chlorella minutissima and Chlorellaemersonii cells transformed with a gene encoding a secreted sucroseinvertase from S. cerevisiae.

FIG. 29 illustrates a gas chromatograph generated by analysis of arenewable diesel product produced in accordance with the methods of thepresent invention, and described in Example 27.

FIG. 30 illustrates a boiling point distribution plot for a renewablediesel product produced in accordance with the methods of the presenceinvention, and described in Example 27.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used with reference to a nucleic acid, “active in microalgae” refersto a nucleic acid that is functional in microalgae. For example, apromoter that has been used to drive an antibiotic resistance gene toimpart antibiotic resistance to a transgenic microalgae is active inmicroalgae. Examples of promoters active in microalgae are promotersendogenous to certain algae species and promoters found in plantviruses.

An “acyl carrier protein” or “ACP” is a protein which binds a growingacyl chain during fatty acid synthesis as a thiol ester at the distalthiol of the 4′-phosphopantetheine moiety and comprises a component ofthe fatty acid synthase complex. The phrase “naturally co-expressed”with reference to an acyl carrier protein in conjunction with a fattyacyl-ACP thioesterase means that the ACP and the thioesterase areco-expressed naturally in a tissue or organism from which they arederived, e.g., because the genes encoding the two enzymes are under thecontrol of a common regulatory sequence or because they are expressed inresponse to the same stimulus.

An “acyl-CoA molecule” or “acyl-CoA” is a molecule comprising an acylmoiety covalently attached to coenzyme A through a thiol ester linkageat the distal thiol of the 4′-phosphopantetheine moiety of coenzyme A.

“Axenic” means a culture of an organism that is free from contaminationby other living organisms.

“Biodiesel” is a biologically produced fatty acid alkyl ester suitablefor use as a fuel in a diesel engine.

The term “biomass” refers to material produced by growth and/orpropagation of cells. Biomass may contain cells and/or intracellularcontents as well as extracellular material. Extracellular materialincludes, but is not limited to, compounds secreted by a cell.

“Bioreactor” means an enclosure or partial enclosure in which cells arecultured, optionally in suspension.

As used herein, a “catalyst” refers to an agent, such as a molecule ormacromolecular complex, capable of facilitating or promoting a chemicalreaction of a reactant to a product without becoming a part of theproduct. A catalyst thus increases the rate of a reaction, after which,the catalyst may act on another reactant to form the product. A catalystgenerally lowers the overall activation energy required for the reactionsuch that it proceeds more quickly or at a lower temperature. Thus areaction equilibrium may be more quickly attained. Examples of catalystsinclude enzymes, which are biological catalysts, heat, which is anon-biological catalyst, and metal catalysts used in fossil oil refiningprocesses.

“Cellulosic material” means the products of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar caner bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

The term “co-culture”, and variants thereof such as “co-cultivate”,refer to the presence of two or more types of cells in the samebioreactor. The two or more types of cells may both be microorganisms,such as microalgae, or may be a microalgal cell cultured with adifferent cell type. The culture conditions may be those that fostergrowth and/or propagation of the two or more cell types or those thatfacilitate growth and/or proliferation of one, or a subset, of the twoor more cells while maintaining cellular growth for the remainder.

The term “cofactor” is used herein to refer to any molecule, other thanthe substrate, that is required for an enzyme to carry out its enzymaticactivity.

As used herein, “complementary DNA” (“cDNA”) is a DNA representation ofmRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or amplification (e.g., via polymerase chain reaction (“PCR”)).

The term “cultivated”, and variants thereof, refer to the intentionalfostering of growth (increases in cell size, cellular contents, and/orcellular activity) and/or propagation (increases in cell numbers viamitosis) of one or more cells by use of intended culture conditions. Thecombination of both growth and propagation may be termed proliferation.The one or more cells may be those of a microorganism, such asmicroalgae. Examples of intended conditions include the use of a definedmedium (with known characteristics such as pH, ionic strength, andcarbon source), specified temperature, oxygen tension, carbon dioxidelevels, and growth in a bioreactor. The term does not refer to thegrowth or propagation of microorganisms in nature or otherwise withoutdirect human intervention, such as natural growth of an organism thatultimately becomes fossilized to produce geological crude oil.

As used herein, the term “cytolysis” refers to the lysis of cells in ahypotonic environment. Cytolysis is caused by excessive osmosis, ormovement of water, towards the inside of a cell (hyperhydration). Thecell cannot withstand the osmotic pressure of the water inside, and soit explodes.

As used herein, the terms “expression vector” or “expression construct”refer to a nucleic acid construct, generated recombinantly orsynthetically, with a series of specified nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. Typically, the expression vector includes a nucleic acid to betranscribed operably linked to a promoter.

“Exogenous gene” refers to a nucleic acid transformed into a cell. Atransformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous) relative to the cell being transformed. Inthe case of a homologous gene, it occupies a different location in thegenome of the cell relative to the endogenous copy of the gene. Theexogenous gene may be present in more than one copy in the cell. Theexogenous gene may be maintained in a cell as an insertion into thegenome or as an episomal molecule.

“Exogenously provided” describes a molecule provided to the culturemedia of a cell culture.

As used herein, a “fatty acyl-ACP thioesterase” is an enzyme thatcatalyzes the cleavage of a fatty acid from an acyl carrier protein(ACP) during lipid synthesis.

As used herein, a “fatty acyl-CoA/aldehyde reductase” is an enzyme thatcatalyzes the reduction of an acyl-CoA molecule to a primary alcohol.

As used herein, a “fatty acyl-CoA reductase” is an enzyme that catalyzesthe reduction of an acyl-CoA molecule to an aldehyde.

As used herein, a “fatty aldehyde decarbonylase” is an enzyme thatcatalyzes the conversion of a fatty aldehyde to an alkane.

As used herein, a “fatty aldehyde reductase” is an enzyme that catalyzesthe reduction of an aldehyde to a primary alcohol.

“Fixed carbon source” means molecule(s) containing carbon, preferablyorganic, that are present at ambient temperature and pressure in solidor liquid form.

“Fungus,” as used herein, means heterotrophic organisms characterized bya chitinous cell wall from the kingdom of fungi.

“Homogenate” means biomass that has been physically disrupted.

As used herein, “hydrocarbon” refers to: (a) a molecule containing onlyhydrogen and carbon atoms wherein the carbon atoms are covalently linkedto form a linear, branched, cyclic, or partially cyclic backbone towhich the hydrogen atoms are attached; or (b) a molecule that onlyprimarily contains hydrogen and carbon atoms and that can be convertedto contain only hydrogen and carbon atoms by one to four chemicalreactions. Nonlimiting examples of the latter include hydrocarbonscontaining an oxygen atom between one carbon and one hydrogen atom toform an alcohol molecule, as well as aldehydes containing a singleoxygen atom. Methods for the reduction of alcohols to hydrocarbonscontaining only carbon and hydrogen atoms are well known. Anotherexample of a hydrocarbon is an ester, in which an organic group replacesa hydrogen atom (or more than one) in an oxygen acid. The molecularstructure of hydrocarbon compounds varies from the simplest, in the formof methane (CH₄), which is a constituent of natural gas, to the veryheavy and very complex, such as some molecules such as asphaltenes foundin crude oil, petroleum, and bitumens. Hydrocarbons may be in gaseous,liquid, or solid form, or any combination of these forms, and may haveone or more double or triple bonds between adjacent carbon atoms in thebackbone. Accordingly, the term includes linear, branched, cyclic, orpartially cyclic alkanes, alkenes, lipids, and paraffin. Examplesinclude propane, butane, pentane, hexane, octane, triolein, andsqualene.

“Hydrocarbon modification enzyme” refers to an enzyme that alters thecovalent structure of a hydrocarbon. Examples of hydrocarbonmodification enzymes include a lipase, a fatty acyl-ACP thioesterase, afatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fattyaldehyde reductase, and a fatty aldehyde decarbonylase. Compoundsproduced by the enzymatic activity of hydrocarbon modification enzymes,including fatty acids, alcohols, aldehydes, alkanes, or other compoundsderived therefrom are referred to herein interchangeably as hydrocarbonsor lipids.

The term “hydrogen:carbon ratio” refers to the ratio of hydrogen atomsto carbon atoms in a molecule on an atom-to-atom basis. The ratio may beused to refer to the number of carbon and hydrogen atoms in ahydrocarbon molecule. For example, the hydrocarbon with the highestratio is methane CH₄ (4:1).

“Hydrophobic fraction” refers to the portion, or fraction, of a materialthat is more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

As used herein, the phrase “increase lipid yield” refers to an increasein the productivity of a microbial culture by, for example, increasingdry weight of cells per liter of culture, increasing the percentage ofcells that constitute lipid, or increasing the overall amount of lipidper liter of culture volume per unit time.

An “inducible promoter” is one that mediates transcription of anoperably linked gene in response to a particular stimulus.

As used herein, the phrase “in operable linkage” refers to a functionallinkage between two sequences, such a control sequence (typically apromoter) and the linked sequence. A promoter is in operable linkagewith an exogenous gene if it can mediate transcription of the gene.

The term “in situ” means “in place” or “in its original position”. Forexample, a culture may contain a first microalgae secreting a catalystand a second microorganism secreting a substrate, wherein the first andsecond cell types produce the components necessary for a particularchemical reaction to occur in situ in the co-culture without requiringfurther separation or processing of the materials.

A “limiting concentration of a nutrient” is a concentration in a culturethat limits the propagation of a cultured organism. A “non-limitingconcentration of a nutrient” is a concentration that supports maximalpropagation during a given culture period. Thus, the number of cellsproduced during a given culture period is lower in the presence of alimiting concentration of a nutrient than when the nutrient isnon-limiting. A nutrient is said to be “in excess” in a culture, whenthe nutrient is present at a concentration greater than that whichsupports maximal propagation.

As used herein, a “lipase” is a water-soluble enzyme that catalyzes thehydrolysis of ester bonds in water-insoluble, lipid substrates. Lipasescatalyze the hydrolysis of lipids into glycerols and fatty acids.

As used herein, a “lipid pathway enzyme” is any enzyme that plays a rolein lipid metabolism, i.e., either lipid synthesis, modification, ordegradation. This term encompasses proteins that chemically modifylipids, as well as carrier proteins.

“Lipids” are a class of hydrocarbon that are soluble in nonpolarsolvents (such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” are a subgroup of lipids called “triacylglycerides.”

As used herein, the term “lysate” refers to a solution containing thecontents of lysed cells.

As used herein, the term “lysis” refers to the breakage of the plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content, often bymechanical, viral or osmotic mechanisms that compromise its integrity.

As used herein, the term “lysing” refers to disrupting the cellularmembrane and optionally the cell wall of a biological organism or cellsufficient to release at least some intracellular content.

“Microalgae” means a eukaryotic microbial organism that contains achloroplast, and optionally that is capable of performingphotosynthesis, or a prokaryotic microbial organism capable ofperforming photosynthesis. Microalgae include obligate photoautotrophs,which cannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of a fixed carbon source.Microalgae can refer to unicellular organisms that separate from sistercells shortly after cell division, such as Chlamydomonas, and can alsorefer to microbes such as, for example, Volvox, which is a simplemulticellular photosynthetic microbe of two distinct cell types.“Microalgae” can also refer to cells such as Chlorella and Dunaliella.“Microalgae” also includes other microbial photosynthetic organisms thatexhibit cell-cell adhesion, such as Agmenellum, Anabaena, andPyrobotrys. “Microalgae” also includes obligate heterotrophicmicroorganisms that have lost the ability to perform photosynthesis,such as certain dinoflagellate algae species.

The terms “microorganism” and “microbe” are used interchangeably hereinto refer to microscopic unicellular organisms.

“Oleaginous yeast,” as used herein, means yeast that can naturallyaccumulate more than 10% of its dry cell weight as lipid or can do so asa result of genetic engineering. Oleaginous yeast includes organismssuch as Yarrowia lipolytica, as well as engineered strains of yeast suchas Saccharomyces cerevisiae that have been engineered to accumulate morethan 10% of their dry cell weight as lipid.

As used herein, the term “osmotic shock” refers to the rupture of cellsin a solution following a sudden reduction in osmotic pressure. Osmoticshock is sometimes induced to release cellular components of such cellsinto a solution.

“Photobioreactor” refers to a container, at least part of which is atleast partially transparent or partially open, thereby allowing light topass through, in which one or more microalgae cells are cultured.Photobioreactors may be closed, as in the instance of a polyethylene bagor Erlenmeyer flask, or may be open to the environment, as in theinstance of an outdoor pond.

As used herein, a “polysaccharide-degrading enzyme” refers to any enzymecapable of catalyzing the hydrolysis, or depolymerization, of anypolysaccharide. For example, cellulases catalyze the hydrolysis ofcellulose.

“Polysaccharides” (also called “glycans”) are carbohydrates made up ofmonosaccharides joined together by glycosidic linkages. Cellulose is anexample of a polysaccharide that makes up certain plant cell walls.Cellulose can be depolymerized by enzymes to yield monosaccharides suchas xylose and glucose, as well as larger disaccharides andoligosaccharides.

“Port”, in the context of a bioreactor, refers to an opening in thebioreactor that allows influx or efflux of materials such as gases,liquids, and cells. Ports are usually connected to tubing leading fromthe photobioreactor.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof an exogenous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

As used herein, the term “renewable diesel” refers to alkanes (such asC:10:0, C12:0, C:14:0, C16:0 and C18:0) produced through hydrogenationand deoxygenation of lipids.

As used herein, the term “sonication” refers to a process of disruptingbiological materials, such as a cell, by use of sound wave energy.

“Species of furfural” refers to 2-Furancarboxaldehyde or a derivativethereof which retains the same basic structural characteristics.

As used herein, “stover” refers to the dried stalks and leaves of a cropremaining after a grain has been harvested.

A “sucrose utilization gene” is a gene that, when expressed, aids theability of a cell to utilize sucrose as an energy source. Proteinsencoded by a sucrose utilization gene are referred to herein as “sucroseutilization enzymes” and include sucrose transporters, sucroseinvertases, and hexokinases such as glucokinases and fructokinases.

“Wastewater” is watery waste which typically contains washing water,laundry waste, faeces, urine and other liquid or semi-liquid wastes. Itincludes some forms of municipal waste as well as secondarily treatedsewage.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

II. General

The invention is premised in part on the insight that certainmicroorganisms can be used to produce oils, fuels, and other hydrocarbonor lipid compositions economically and in large quantities for use inthe transportation fuel, petrochemical industry, and/or food andcosmetic industries, among other applications. Suitable microorganismsinclude microalgae, oleaginous yeast, and fungi. A preferred genus ofmicroalgae for use in the invention is the lipid-producing microalgaeChlorella. Transesterification of lipids yields long-chain fatty acidesters useful as biodiesel. Other enzymatic and chemical processes canbe tailored to yield fatty acids, aldehydes, alcohols, alkanes, andalkenes. The present application describes methods for geneticmodification of multiple species and strains of microorganisms,including Chlorella and similar microbes to provide organisms havingcharacteristics that facilitate the production of lipid suitable forconversion into oils, fuels, and oleochemicals. In some applications,renewable diesel, jet fuel, or other hydrocarbon compounds are produced.The present application also describes methods of cultivating microalgaefor increased productivity and increased lipid yield, and/or for morecost-effective production of the compositions described herein.

In particular embodiments, the present application describes geneticallyengineering strains of microalgae with one or more exogenous genes. Forexample, microalgae that produce high levels of triacylglycerides (TAGs)suitable for biodiesel can be engineered to express a lipase, which canfacilitate transesterification of microalgal TAGs. The lipase canoptionally be expressed using an inducible promoter, so that the cellscan first be grown to a desirable density in a fermentor and thenharvested, followed by induction of the promoter to express the lipase,optionally in the presence of sufficient alcohol to drive conversion ofTAGs to fatty acid esters.

Some microalgal lipid is sequestered in cell membranes and othernon-aqueous parts of the cell. Therefore, to increase the yield of thetransesterification reaction, it can be beneficial to lyse the cells toincrease the accessibility of the lipase to the lipid. Cell disruptioncan be performed, for example, mechanically, through addition ofpressurized steam, or by employing a virus that lyses the microalgaecells, expressing a gene to produce a lytic protein in the cell, ortreating the culture with an agent that lyses microalgae cells. Steamtreatment of microalgae for cell disruption is described, for example,in U.S. Pat. No. 6,750,048.

Also disclosed herein is the genetic engineering of microalgae thatproduce high levels of TAGs to express a gene that lyses microalgaecells, such as for example, a gene from a lytic virus. This gene can beexpressed using an inducible promoter, so that the cells can first begrown to a desirable density in a fermentor and then harvested, followedby induction of the promoter to express the gene to lyse the cells. Agene encoding a polysaccharide-degrading enzyme, for example, can beexpressed to lyse the cells.

Optionally, the lipase can be expressed in an intracellular compartment,where it remains separate from the majority of the microalgal lipiduntil transesterification. Generally, it is preferable to carry outtransesterification after water has been substantially removed from thepreparation and/or an excess of alcohol has been added. Lipases can usewater, as well as alcohol, as a substrate in transesterification. Withwater, the lipid is conjugated to a hydroxyl moiety to produce a polarfatty acid, rather than an ester. With an alcohol, such as methanol, thelipid is conjugated to a methyl group, producing a non-polar fatty acidester, which is typically preferable for a transportation fuel. To limitexposure of the lipase to microalgal lipid until conditions are suitablefor transesterification to produce fatty acid esters, the lipase can beexpressed, for example, in the chloroplast, mitochondria, or othercellular organelle. This compartmentalized expression results insequestration of the lipase from the majority of the cellular lipiduntil after the cells have been disrupted.

In other particular embodiments, the present application describesgenetically engineering strains of microalgae, oleaginous yeast,bacteria, or fungi with one or more exogenous genes to produce varioushydrocarbon compounds. For example, microalgae that would naturally, orthrough genetic modification, produce high levels of lipids can beengineered (or further engineered) to express an exogenous fattyacyl-ACP thioesterase, which can facilitate the cleavage of fatty acidsfrom the acyl carrier protein (ACP) during lipid synthesis. These fattyacids can be recovered or, through further enzymatic processing withinthe cell, yield other hydrocarbon compounds. Optionally, the fattyacyl-ACP thioesterase can be expressed from a gene operably linked to aninducible promoter, and/or can be expressed in an intracellularcompartment.

The fatty acyl-ACP thioesterase can be chosen based on its specificityfor a growing (during fatty acid synthesis) fatty acid having aparticular carbon chain length. For example, the fatty acyl-ACPthioesterase can have a specificity for a carbon chain length rangingfrom 8 to 34 carbon atoms, preferably from 8 to 18 carbon atoms, andmore preferably from 10 to 14 carbon atoms. A specificity for a fattyacid with 12 carbon atoms is most preferred.

Further, the invention provides genetically engineered strains ofmicroalgae to express two or more exogenous genes, such as, for example,a lipase and a lytic gene, e.g., one encoding a polysaccharide-degradingenzyme. One or both genes can be expressed using an inducible promoter,which allows the relative timing of expression of these genes to becontrolled to enhance the lipid yield and conversion to fatty acidesters. The invention also provides vectors and methods for engineeringlipid-producing microbes to metabolize sucrose, which is an advantageoustrait because it allows the engineered cells to convert sugar cane orother feedstocks into lipids appropriate for production of oils, fuels,oleochemicals and the like.

In other embodiments, the invention provides genetically engineeredstrains of microbes (e.g., microalgae, oleaginous yeast, bacteria, orfungi) that express two or more exogenous genes, such as, for example, afatty acyl-ACP thioesterase and a fatty acyl-CoA/aldehyde reductase, thecombined action of which yields an alcohol product. The inventionfurther provides other combinations of exogenous genes, includingwithout limitation, a fatty acyl-ACP thioesterase and a naturallyco-expressed acyl carrier protein to generate length-specific fattyacids, or a fatty acyl-ACP thioesterase and a fatty acyl-CoA reductaseto generate aldehydes. The invention also provides for the combinationof a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and afatty aldehyde decarbonylase to generate alkanes or alkenes. One or moreof the exogenous genes can be expressed using an inducible promoter.

The invention provides further modifications of microalgae, for exampleto provide microalgae with desired growth characteristics and/or toenhance the amount and/or quality of lipids produced. For example,microalgae can be engineered to increase carbon flux into the lipidpathway and/or modify the lipid pathway to beneficially alter theproportions or properties of lipid produced by the cells.

This application discloses genetically engineering strains of microalgaeto express two or more exogenous genes, one encoding a transporter of afixed carbon source (such as sucrose) and a second encoding a sucroseinvertase enzyme. The resulting fermentable organisms producehydrocarbons at lower manufacturing cost than what has been obtainableby previously known methods of biological hydrocarbon production. Theinsertion of the two exogenous genes described above can be combinedwith the disruption of polysaccharide biosynthesis through directedand/or random mutagenesis, which steers ever greater carbon flux intohydrocarbon production. Individually and in combination, trophicconversion, engineering to alter hydrocarbon production and treatmentwith exogenous enzymes alter the hydrocarbon composition produced by amicroorganism. The alteration can be a change in the amount ofhydrocarbons produced, the amount of one or more hydrocarbon speciesproduced relative to other hydrocarbons, and/or the types of hydrocarbonspecies produced in the microorganism. For example, microalgae can beengineered to produce a higher amount and/or percentage of TAGs.

III. Oil- or Lipid-Producing Microorganisms

Any species of organism that produces suitable lipid or hydrocarbon canbe used, although microorganisms that naturally produce high levels ofsuitable lipid or hydrocarbon are preferred. Production of hydrocarbonsby microorganisms is reviewed by Metzger et al. Appl MicrobiolBiotechnol (2005) 66: 486-496 and A Look Back at the U.S. Department ofEnergy's Aquatic Species Program: Biodiesel from Algae,NREL/TP-580-24190, John Sheehan, Terri Dunahay, John Benemann and PaulRoessler (1998).

Considerations affecting the selection of microorganisms for use in theinvention include, in addition to production of suitable lipids orhydrocarbons for production of oils, fuels, and oleochemicals: (1) highlipid content as a percentage of cell weight; (2) ease of growth; (3)ease of genetic engineering; and (4) ease of biomass processing. Inparticular embodiments, the wild-type or genetically engineeredmicroorganism yields cells that are at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, or at least 70% or morelipid. Preferred organisms grow heterotrophically (on sugars in theabsence of light) or can be engineered to do so using, for example,methods disclosed herein. The ease of transformation and availability ofselectable markers and promoters, constitutive and/or inducible, thatare functional in the microorganism affect the ease of geneticengineering. Processing considerations can include, for example, theavailability of effective means for lysing the cells.

A. Algae

In one embodiment of the present invention, the microorganism is amicroalgae. Nonlimiting examples of microalgae that can be used inaccordance with the present invention can be found in Table 1.

TABLE 1 Examples of microalgae. Achnanthes orientalis, Agmenellum,Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis linea,Amphora coffeiformis punctata, Amphora coffeiformis taylori, Amphoracoffeiformis tenuis, Amphora delicatissima, Amphora delicatissimacapitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Bracteococcus minor, Bracteococcusmedionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri,Chaetoceros muelleri subsalsum, Chaetoceros sp., Chlorella anitrata,Chlorella Antarctica, Chlorella aureoviridis, Chlorella candida,Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea,Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata,Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var.actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri,Chlorella lobophora (strain SAG 37.88), Chlorella luteoviridis,Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.lutescens, Chlorella miniata, Chlorella minutissima, Chlorellamutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva,Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides(including any of UTEX strains 1806, 411, 264, 256, 255, 250, 249, 31,29, 25), Chlorella protothecoides var. acidicola, Chlorella regularis,Chlorella regularis var. minima, Chlorella regularis var. umbricata,Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophilavar. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorellasorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorellavulgaris f. tertia, Chlorella vulgaris var. autotrophica, Chlorellavulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorellavulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f.viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorellatrebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcumsp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotellameneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil,Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime,Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliellaprimolecta, Dunaliella salina, Dunaliella terricola, Dunaliellatertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaeraviridis, Eremosphaera sp., Ellipsoidon sp., Euglena, Franceia sp.,Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnionsp., Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana,Lepocinclis, Micractinium, Micractinium (UTEX LB 2614), Monoraphidiumminutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina,Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Naviculapseudotenelloides, Navicula pelliculosa, Navicula saprophila, Naviculasp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschiaalexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii,Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis,Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp.,Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmusarmatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcussp., Synechococcus sp., Tetraedron, Tetraselmis sp., Tetraselmissuecica, Thalassiosira weissflogii, and Viridiella fridericiana

1. Chlorella

In a preferred embodiment of the present invention, the microorganism isof the genus Chlorella, preferably, Chlorella protothecoides, Chlorellaellipsoidea, Chlorella minutissima, or Chlorella emersonii.

Chlorella is a genus of single-celled green algae, belonging to thephylum Chlorophyta. It is spherical in shape, about 2 to 10 μm indiameter, and is without flagella. Some species of Chlorella arenaturally heterotrophic.

Chlorella, particularly Chlorella protothecoides, is a preferredmicroorganism for use in the invention because of its high compositionof lipid, particularly long-chain lipid suitable for biodiesel. Inaddition, this microalgae grows heterotrophically and can be geneticallyengineered as demonstrated in the Examples herein.

In a preferred embodiment of the present invention, the microorganismused for expression of a transgene is of the genus Chlorella,preferably, Chlorella protothecoides, Chlorella minutissima, orChlorella emersonii. Examples of expression of transgenes in, e.g.,Chlorella, can be found in the literature (see for example CurrentMicrobiology Vol. 35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao.2000 July; 16(4):443-6; Current Microbiology Vol. 38 (1999), pp.335-341; Appl Microbiol Biotechnol (2006) 72: 197-205; MarineBiotechnology 4, 63-73, 2002; Current Genetics 39:5, 365-370 (2001);Plant Cell Reports 18:9, 778-780, (1999); Biologia Plantarium 42(2):209-216, (1999); Plant Pathol. J 21(1): 13-20, (2005)). Also seeExamples herein. Other lipid-producing microalgae can be engineered aswell, including prokaryotic Microalgae (see Kalscheuer et al., AppliedMicrobiology and Biotechnology, Volume 52, Number 4/October, 1999).

2. Identification of Chlorella Species

Species of Chlorella for use in the invention can be identified byamplification of certain target regions of the genome. For example,identification of a specific Chlorella species or strain can be achievedthrough amplification and sequencing of nuclear and/or chloroplast DNAusing primers and methodology using any region of the genome, forexample using the methods described in Wu et al., Bot. Bull. Acad. Sin.(2001) 42:115-121 Identification of Chlorella spp. isolates usingribosomal DNA sequences. Well established methods of phylogeneticanalysis, such as amplification and sequencing of ribosomal internaltranscribed spacer (ITS1 and ITS2 rDNA), 18S rRNA, and other conservedgenomic regions can be used by those skilled in the art to identifyspecies of not only Chlorella, but other hydrocarbon and lipid producingorganisms capable of using the methods disclosed herein. For examples ofmethods of identification and classification of algae also see forexample Genetics, 2005 August; 170(4): 1601-10 and RNA, 2005 April;11(4):361-4.

B. Oleaginous Yeast

In one embodiment of the present invention, the microorganism is anoleaginous yeast. Nonlimiting examples of oleaginous yeast that can beused in accordance with the present invention can be found in Table 2.

TABLE 2 Examples of oleaginous yeast. Cryptococcus curvatus,Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyceslipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorulagracilis, and Yarrowia lipolytica

C. Other Fungi

In one embodiment of the present invention, the microorganism is afungus. Nonlimiting examples of fungi that can be used in accordancewith the present invention can be found in Table 3.

TABLE 3 Examples of fungi. Mortierella, Mortierrla vinacea, Mortierellaalpine, Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus,Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium,Cladosporium, Malbranchea, Rhizopus, and Pythium

D. Bacteria

In one embodiment of the present invention, the microorganism is abacterium.

Examples of expression of exogenous genes in bacteria, such as E. coli,are well known; see for example Molecular Cloning: A Laboratory Manual,Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press).

IV. Methods of Culturing Microorganisms

Microorganisms are cultured both for purposes of conducting geneticmanipulations and for subsequent production of hydrocarbons (e.g.,lipids, fatty acids, aldehydes, alcohols, and alkanes). The former typeof culture is conducted on a small scale and initially, at least, underconditions in which the starting microorganism can grow. For example, ifthe starting microorganism is a photoautotroph the initial culture isconducted in the presence of light. The culture conditions can bechanged if the microorganism is evolved or engineered to growindependently of light. Culture for purposes of hydrocarbon productionis usually conducted on a large scale. Preferably a fixed carbon sourceis present. The culture can also be exposed to light some or all of thetime.

Microalgae can be cultured in liquid media. The culture can be containedwithin a bioreactor. Optionally, the bioreactor does not allow light toenter. Alternatively, microalgae can also be cultured inphotobioreactors that contain a fixed carbon source and allow light tostrike the cells. Exposure of microalgae cells to light, even in thepresence of a fixed carbon source that the cells transport and utilize(i.e., mixotrophic growth), nonetheless accelerates growth compared toculturing cells in the dark. Culture condition parameters can bemanipulated to optimize total hydrocarbon production, the combination ofhydrocarbon species produced, and/or production of a hydrocarbonspecies. In some instances it is preferable to culture cells in thedark, such as, for example, when using extremely large (40,000 liter andhigher) fermentors that do not allow light to strike the culture.

Microalgal culture media typically contains components such as a fixednitrogen source, trace elements, optionally a buffer for pH maintenance,and phosphate. Other components can include a fixed carbon source suchas acetate or glucose, and salts such as sodium chloride, particularlyfor seawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂O and(NH₄)₆Mo₇O₂₄.4H₂O.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, glucose, fructose, sucrose, galactose,xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside,and/or glucuronic acid. The one or more carbon source(s) can be suppliedat a concentration of at least about 50 μM, at least about 100 μM, atleast about 500 μM, at least about 5 mM, at least about 50 mM, and atleast about 500 mM, of one or more exogenously provided fixed carbonsource(s). Some microalgae species can grow by utilizing a fixed carbonsource such as glucose or acetate in the absence of light. Such growthis known as heterotrophic growth. For Chlorella protothecoides, forexample, heterotrophic growth results in high production of biomass andaccumulation of high lipid content in cells.

Some microorganisms naturally grow on or can be engineered to grow on afixed carbon source that is a heterogeneous source of compounds such asmunicipal waste, secondarily treated sewage, wastewater, and othersources of fixed carbon and other nutrients such as sulfates,phosphates, and nitrates. The sewage component serves as a nutrientsource in the production of hydrocarbons, and the culture provides aninexpensive source of hydrocarbons.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements andother media constituents.

A. Photosynthetic Growth

Microalgae can be grown in the presence of light. The number of photonsstriking a culture of microalgae cells can be manipulated, as well asother parameters such as the wavelength spectrum and ratio of dark:lighthours per day. Microalgae can also be cultured in natural light, as wellas simultaneous and/or alternating combinations of natural light andartificial light. For example, microalgae of the genus Chlorella can becultured under natural light during daylight hours and under artificiallight during night hours.

The gas content of a photobioreactor to grow microorganisms likemicroalgae can be manipulated. Part of the volume of a photobioreactorcan contain gas rather than liquid. Gas inlets can be used to pump gasesinto the photobioreactor. Any gas can be pumped into a photobioreactor,including air, air/CO₂ mixtures, noble gases such as argon and others.The rate of entry of gas into a photobioreactor can also be manipulated.Increasing gas flow into a photobioreactor increases the turbidity of aculture of microalgae. Placement of ports conveying gases into aphotobioreactor can also affect the turbidity of a culture at a givengas flow rate. Air/CO₂ mixtures can be modulated to generate optimalamounts of CO₂ for maximal growth by a particular organism. Microalgaegrow significantly faster in the light under, for example, 3% CO₂/97%air than in 100% air. 3% CO₂/97% air is approximately 100-fold more CO₂than found in air. For example, air:CO₂ mixtures of about 99.75%air:0.25% CO₂, about 99.5% air:0.5% CO₂, about 99.0% air:1.00% CO₂,about 98.0% air:2.0% CO₂, about 97.0% air:3.0% CO₂, about 96.0% air:4.0%CO₂, and about 95.00% air:5.0% CO₂ can be infused into a bioreactor orphotobioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and impellers, rocking of a culture, stir bars,infusion of pressurized gas, and other instruments.

Photobioreactors can have ports allowing entry of gases, solids,semisolids and liquids into the chamber containing the microalgae. Portsare usually attached to tubing or other means of conveying substances.Gas ports, for example, convey gases into the culture. Pumping gasesinto a photobioreactor can serve to both feed cells CO₂ and other gasesand to aerate the culture and therefore generate turbidity. The amountof turbidity of a culture varies as the number and position of gas portsis altered. For example, gas ports can be placed along the bottom of acylindrical polyethylene bag. Microalgae grow faster when CO₂ is addedto air and bubbled into a photobioreactor. For example, a 5% CO₂:95% airmixture is infused into a photobioreactor containing Botryococcus cells(see for example J Agric Food Chem. 2006 Jun. 28; 54(13):4593-9; JBiosci Bioeng. 1999; 87(6):811-5; and J Nat Prod. 2003 June;66(6):772-8).

Photobioreactors can be exposed to one or more light sources to providemicroalgae with light as an energy source via light directed to asurface of the photobioreactor. Preferably the light source provides anintensity that is sufficient for the cells to grow, but not so intenseas to cause oxidative damage or cause a photoinhibitive response. Insome instances a light source has a wavelength range that mimics orapproximately mimics the range of the sun. In other instances adifferent wavelength range is used. Photobioreactors can be placedoutdoors or in a greenhouse or other facility that allows sunlight tostrike the surface. Preferred photon intensities for species of thegenus Botryococcus are between 25 and 500 μE m⁻² s⁻¹ (see for examplePhotosynth Res. 2005 June; 84(1-3):21-7).

Photobioreactors preferably have one or more ports that allow mediaentry. It is not necessary that only one substance enter or leave aport. For example, a port can be used to flow culture media into thephotobioreactor and then later can be used for sampling, gas entry, gasexit, or other purposes. In some instances a photobioreactor is filledwith culture media at the beginning of a culture and no more growthmedia is infused after the culture is inoculated. In other words, themicroalgal biomass is cultured in an aqueous medium for a period of timeduring which the microalgae reproduce and increase in number; howeverquantities of aqueous culture medium are not flowed through thephotobioreactor throughout the time period. Thus in some embodiments,aqueous culture medium is not flowed through the photobioreactor afterinoculation.

In other instances culture media can be flowed though thephotobioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments media is infusedinto the photobioreactor after inoculation but before the cells reach adesired density. In other words, a turbulent flow regime of gas entryand media entry is not maintained for reproduction of microalgae until adesired increase in number of said microalgae has been achieved.

Photobioreactors preferably have one or more ports that allow gas entry.Gas can serve to both provide nutrients such as CO₂ as well as toprovide turbulence in the culture media. Turbulence can be achieved byplacing a gas entry port below the level of the aqueous culture media sothat gas entering the photobioreactor bubbles to the surface of theculture. One or more gas exit ports allow gas to escape, therebypreventing pressure buildup in the photobioreactor. Preferably a gasexit port leads to a “one-way” valve that prevents contaminatingmicroorganisms from entering the photobioreactor. In some instancescells are cultured in a photobioreactor for a period of time duringwhich the microalgae reproduce and increase in number, however aturbulent flow regime with turbulent eddies predominantly throughout theculture media caused by gas entry is not maintained for all of theperiod of time. In other instances a turbulent flow regime withturbulent eddies predominantly throughout the culture media caused bygas entry can be maintained for all of the period of time during whichthe microalgae reproduce and increase in number. In some instances apredetermined range of ratios between the scale of the photobioreactorand the scale of eddies is not maintained for the period of time duringwhich the microalgae reproduce and increase in number. In otherinstances such a range can be maintained.

Photobioreactors preferably have at least one port that can be used forsampling the culture. Preferably a sampling port can be used repeatedlywithout altering compromising the axenic nature of the culture. Asampling port can be configured with a valve or other device that allowsthe flow of sample to be stopped and started. Alternatively a samplingport can allow continuous sampling. Photobioreactors preferably have atleast one port that allows inoculation of a culture. Such a port canalso be used for other purposes such as media or gas entry.

B. Heterotrophic Growth

As an alternative to photosynthetic growth of microorganisms, asdescribed above, some microorganisms can be cultured under heterotrophicgrowth conditions in which a fixed carbon source provides energy forgrowth and lipid accumulation.

In one heterotrophic culture method in accordance with the invention,the cost of biodiesel production, crude, partially purified, or purifiedglycerol produced as a byproduct of lipid transesterification can beemployed as a feedstock for fermenting, for example, lipid-producingmicrobial cultures. Thus, the invention encompasses culturing a microbe(e.g., a microalgae) in a first microbial culture; recovering microbiallipid from the culture; subjecting the microbial lipid totransesterification to produce fatty acid ester(s) and glycerol, asdescribed above; and adding the glycerol to a second microbial cultureas a feedstock. The first and second microbial cultures can, but neednot, be cultures of the same microbe. If desired, a continuous systemcan be devised whereby glycerol produced from the lipid recovered from aculture can be fed back into the same culture.

The invention provides significantly improved culture parametersincorporating the use of glycerol for fermentation of multiple genera ofboth eukaryotic and prokaryotic microbes, including microbes of thegenera Chlorella, Navicula, Scenedesmus, and Spirulina. As the Examplesdemonstrate, microbes of extremely divergent evolutionary lineages,including Chlorella, Navicula, Scenedesmus, and Spirulina as well ascultures of multiple distinct Chlorella species and strains grow verywell on not only purified reagent-grade glycerol, but also on acidulatedand non-acidulated glycerol byproduct from biodieseltransesterification. In some instances microalgae, such as Chlorellastrains, undergo cell division faster in the presence of glycerol thanin the presence of glucose. In these instances, two-stage growthprocesses in which cells are first fed glycerol to rapidly increase celldensity, and are then fed glucose to accumulate lipids can improve theefficiency with which lipids are produced. The use of the glycerolbyproduct of the transesterification process provides significanteconomic advantages when put back into the production process. Otherfeeding methods are provided as well, such as mixtures of glycerol andglucose. Feeding such mixtures also captures the same economic benefits.In addition, the invention provides methods of feeding alternativesugars to microalgae such as sucrose in various combinations withglycerol. These benefits provided by the invention have beendemonstrated herein on microbes from extremely divergent evolutionarylineages, including both prokaryotes and eukaryotes, demonstrating theutility of the invention for microbial fermentation.

Standard methods for the growth and propagation of Chlorellaprotothecoides are known (see for example Miao and Wu, J. Biotechnology,2004, 11:85-93 and Miao and Wu, Biosource Technology (2006) 97:841-846).The invention also provides novel growth conditions for Chlorella. Forexample, multiple species of Chlorella and multiple strains within aspecies can be grown in the presence of glycerol, including glycerolbyproduct from biodiesel transesterification.

For hydrocarbon production, cells, including recombinant cells of theinvention described herein, are preferably cultured or fermented inlarge quantities. The culturing may be in large liquid volumes, such asin suspension cultures as an example. Other examples include startingwith a small culture of cells which expand into a large biomass incombination with cell growth and propagation as well as hydrocarbonproduction. Bioreactors or steel fermentors can be used to accommodatelarge culture volumes. A fermentor similar those used in the productionof beer and/or wine is suitable, as are extremely large fermentors usedin the production of ethanol.

Appropriate nutrient sources for culture in a fermentor are provided.These include raw materials such as one or more of the following: afixed carbon source such as glucose, corn starch, depolymerizedcellulosic material, sucrose, sugar cane, sugar beet, lactose, milkwhey, or molasses; a fat source, such as fats or vegetable oils; anitrogen source, such as protein, soybean meal, cornsteep liquor,ammonia (pure or in salt form), nitrate or nitrate salt, or molecularnitrogen; and a phosphorus source, such as phosphate salts.Additionally, a fermentor allows for the control of culture conditionssuch as temperature, pH, oxygen tension, and carbon dioxide levels.Optionally, gaseous components, like oxygen or nitrogen, can be bubbledthrough a liquid culture. Other Starch (glucose) sources such as wheat,potato, rice, and sorghum. Other carbon sources include process streamssuch as technical grade glycerol, black liquor, organic acids such asacetate, and molasses. Carbon sources can also be provided as a mixture,such as a mixture of sucrose and depolymerized sugar beet pulp.

A fermentor can be used to allow cells to undergo the various phases oftheir growth cycle. As an example, an inoculum of hydrocarbon-producingcells can be introduced into a medium followed by a lag period (lagphase) before the cells begin growth. Following the lag period, thegrowth rate increases steadily and enters the log, or exponential,phase. The exponential phase is in turn followed by a slowing of growthdue to decreases in nutrients and/or increases in toxic substances.After this slowing, growth stops, and the cells enter a stationary phaseor steady state, depending on the particular environment provided to thecells.

Hydrocarbon production by cells disclosed herein can occur during thelog phase or thereafter, including the stationary phase whereinnutrients are supplied, or still available, to allow the continuation ofhydrocarbon production in the absence of cell division.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 20% by weight of lipid,preferably at least about 40% by weight, more preferably at least about50% by weight, and most preferably at least about 60% by weight.

A surprising discovery is that multiple species, and multiple strainswithin a species of Chlorella perform better in the presence of glycerolbyproduct from transesterification than in an equivalent amount ofreagent grade glycerol. Glycerol byproduct from transesterificationusually contains residual methanol and other contaminants in addition toglycerol. For example, FIGS. 1-6 demonstrate that strains of Chlorellaprotothecoides and Chlorella kessleri exhibit better productivity onacidulated and non-acidulated glycerol byproduct from lipidtransesterification reactions than when grown on pure reagent gradeglycerol. Other microbes, such as Scenedesmus and Navicula microalgaecan also perform better in the presence of glycerol byproduct fromtransesterification than in an equivalent amount of reagent gradeglycerol.

Dry Cell Weight per Liter: FIG. 1 demonstrates that dry cell weight washigher on biodiesel glycerol byproduct than on pure glycerol, and thistrend held true when the cells were grown in glycerol by itself or incombination with glucose. FIG. 2 shows the same trends with additionalstrains of Chlorella. FIG. 12( b) demonstrates that dry cell weight perliter of Scenedesmus armatus is higher on acidulated and non-acidulatedbiodiesel byproducts glycerol than on pure reagent grade glycerol. FIG.13 demonstrates that dry cell weight per liter of Navicula pelliculosais higher on non-acidulated biodiesel byproduct glycerol than on purereagent grade glycerol.

Lipid Content per liter: FIGS. 3 and 4 demonstrates that with multiplespecies of Chlorella and multiple strains within a species of Chlorella,lipid levels per liter are higher when the cells are cultured in thepresence of biodiesel glycerol byproduct than when cultured in thepresence of equivalent concentrations of pure reagent grade glycerol.

Lipid as a Percentage of Cell Weight: FIGS. 5 and 6 demonstrate thatmultiple species of Chlorella and multiple strains within a species ofChlorella accumulate a higher percentage of dry cell weight as lipidwhen cultured in the presence of biodiesel glycerol byproduct than whencultured in the presence of equivalent concentrations of pure reagentgrade glycerol. FIG. 11 demonstrates that both Spirulina platensis andNavicula pelliculosa can accumulate a higher percentage of dry cellweight as lipid when cultured in the presence of biodiesel glycerolbyproduct than when cultured in the presence of equivalentconcentrations of pure reagent grade glycerol. FIG. 12( a) demonstratesthat Scenedesmus armatus can accumulate a higher percentage of dry cellweight as lipid when cultured in the presence of biodiesel glycerolbyproduct than when cultured in the presence of equivalentconcentrations of pure reagent grade glycerol.

Another surprising result is that multiple species of microbes,including microalgae such as Chlorella and multiple strains within aspecies of Chlorella, and other microalgae such as Scenedesmus,Navicula, and Spirulina exhibit better characteristics as biodieselproducers in the presence of mixtures of glycerol and glucose than inthe presence of only glucose.

Lipid Content per liter: FIG. 7 demonstrates that Chlorella canaccumulate higher levels of lipid per liter of culture in the presenceof 1% glycerol/1% glucose than in the presence of 2% glucose.

Dry Cell Weight per Liter: FIG. 12( b) demonstrates that dry cell weightper liter of Scenedesmus armatus is higher when cultured in the presenceof 1% biodiesel byproduct glycerol/1% glucose than in the presence of 2%glucose. FIG. 13 demonstrates that dry cell weight per liter of Naviculapelliculosa is higher when cultured in the presence of 1% biodieselbyproduct glycerol/1% glucose than in the presence of 2% glucose.

Lipid as a Percentage of Cell Weight: FIG. 8 demonstrates that Chlorellacan accumulate a higher percentage of dry cell weight as lipid whencultured in the presence of an equal concentration (weight percent)mixture of glycerol and glucose than when cultured in the presence ofonly glucose. FIG. 11( a) demonstrates that Spirulina platensis canaccumulate a higher percentage of dry cell weight as lipid when culturedin the presence of an equal concentration (weight percent) mixture ofbiodiesel byproduct glycerol and glucose than when cultured in thepresence of only glucose. FIG. 11( b) demonstrates that Naviculapelliculosa can accumulate a higher percentage of dry cell weight aslipid when cultured in the presence of an equal concentration (weightpercent) mixture of reagent grade glycerol and glucose, as well asbiodiesel byproduct glycerol and glucose, than when cultured in thepresence of only glucose. FIG. 12( b) demonstrates that Scenedesmusarmatus can accumulate a higher percentage of dry cell weight as lipidwhen cultured in the presence of an equal concentration (weight percent)mixture of biodiesel byproduct glycerol and glucose than when culturedin the presence of only glucose.

An additional and unexpected discovery is that adding glycerol andglucose to microbes, including microalgae such as Chlorella,Scenedesmus, and Navicula sequentially rather than as a single batchmixture of glycerol and glucose can generate additional yield gains.This attribute of multiple species of Chlorella and multiple strainswithin a species of Chlorella was tested in the presence of bothbiodiesel glycerol byproduct and reagent grade glycerol.

Lipid as a Percentage of Cell Weight: FIG. 8 demonstrates that Chlorellacan accumulate a higher percentage of dry cell weight as lipid whenglycerol is added to a culture for a first period of time, followed byaddition of glucose and continued culturing for a second period of time,than when the same quantities of glycerol and glucose are added togetherat the beginning of the experiment.

Lipid Content per liter: FIG. 9 shows Chlorella exhibiting higher levelsof lipid per liter of culture in when glycerol and glucose are addedsequentially than when the same quantities of glycerol and glucose areadded together at the beginning of the experiment. This trend wasobserved when acidulated biodiesel byproduct glycerol, non-acidulatedbiodiesel byproduct glycerol, or reagent grade glycerol was used.

Dry Cell Weight per Liter: FIG. 10 demonstrates four different strainsof Chlorella of two different species accumulating higher dry cellweight per liter of culture when glycerol and glucose are addedsequentially than when the same quantities of glycerol and glucose areadded together at the beginning of the experiment. This trend wasobserved when acidulated biodiesel byproduct glycerol, non-acidulatedbiodiesel byproduct glycerol, or reagent grade glycerol was used. FIGS.14( a) and (b) demonstrates that both Scenedesmus armatus and Naviculapelliculosa can exhibit increases in dry cell weight per liter whenbiodiesel byproduct glycerol only is added to a culture for a firstperiod of time, followed later by addition of glucose, compared toadding identical amounts of glycerol and glucose at the beginning of thefermentation.

Three different markers of productivity (dry cell weight per liter,grams per liter of lipid, and percentage of dry cell weight as lipid) inmicrobial lipid production are improved by the use of biodieselbyproduct and temporal separation of carbon sources. The inventiontherefore provides novel methods of generating higher quantities oflipid per unit time in multiple species of microbes from highlydivergent areas of the evolutionary tree, including both prokaryotes andeukaryotes. The methods of manufacturing lipids and hydrocarbonsdisclosed herein using glycerol are not limited to microalgae, but canbe used with any microbe capable of utilizing glycerol as an energysource.

In an alternate heterotrophic growth method in accordance with thepresent invention, microorganisms can be cultured using depolymerizedcellulosic biomass as a feedstock. Cellulosic biomass (e.g., stover,such as corn stover) is inexpensive and readily available; however,attempts to use this material as a feedstock for yeast have failed. Inparticular, such feedstock have been found to be inhibitory to yeastgrowth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemi-cellulose). By contrast,microalgae can grow on processed cellulosic material. Accordingly, theinvention provides a method of culturing a microalgae in the presence ofa cellulosic material and/or a 5-carbon sugar. Cellulosic materialsgenerally include:

Component Percent Dry Weight Cellulose 40-60% Hemicellulose 20-40%Lignin 10-30%

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

Surprisingly, some species of Chlorella have been shown herein toexhibit higher levels of productivity when cultured on a combination ofglucose and xylose than when cultured on either glucose or xylose alone.This synergistic effect provides a significant advantage in that itallows cultivation of Chlorella on combinations of xylose and glucose,such as cellulosic material, and is shown in FIG. 15.

In still another alternative heterotrophic growth method in accordancewith the present invention, which itself may optionally be used incombination with the methods described above, sucrose, produced byexample from sugar cane or sugar beet, is used as a feedstock. Asdescribed in greater detail in the section entitled “MicrobeEngineering” below, lipid production can be facilitated or made moreefficient through the engineering of microbes such as Chlorella, toutilize sucrose as a carbon source. For example, expression of a sucrosetransporter and a sucrose invertase allows Chlorella to transportsucrose into the cell from the culture media and hydrolyze sucrose toyield glucose and fructose. Optionally, a fructokinase can be expressedas well in instances where endogenous hexokinase activity isinsufficient for maximum phosphorylation of fructose. Examples ofsuitable sucrose transporters are Genbank accession numbers CAD91334,CAB92307, and CAA53390. Examples of suitable sucrose invertases areGenbank accession numbers CAB95010, NP_(—)012104 and CAA06839. Examplesof suitable fructokinases are Genbank accession numbers P26984, P26420and CAA43322. Vectors for transformation of microalgae, includingChlorella, encoding one or more of such genes can be designed asdescribed herein.

Secretion of a sucrose invertase can obviate the need for expression ofa transporter that can transport sucrose into the cell. This is becausea secreted invertase catalyzes the conversion of a molecule of sucroseinto a molecule of glucose and a molecule of fructose, both of which canbe transported and utilized by microbes disclosed herein. For example,expression of a sucrose invertase (such as SEQ ID NO:14) with asecretion signal (such as that of SEQ ID NO:15 (from yeast), SEQ IDNO:16 (from higher plants), SEQ ID NO:17 (eukaryotic consensus secretionsignal), and SEQ ID NO:18 (combination of signal sequence from higherplants and eukaryotic consensus) generates invertase activity outsidethe cell. See Hawkins et al., Current Microbiology Vol. 38 (1999), pp.335-341 for examples of secretion signals active in Chlorella.Expression of such a protein, as enabled by the genetic engineeringmethodology disclosed herein, allows cells already capable of utilizingextracellular glucose as an energy source to utilize sucrose as anextracellular energy source. In cells such as Chlorella protothecoides,Chlorella minutissima, and Chlorella emersonii which as demonstratedherein can use both extracellular fructose and extracellular glucose asan energy source, secretion of an invertase can provide the solecatalytic activity necessary for use of sucrose as an efficient,inexpensive energy source.

For example, as shown in FIG. 26, Chlorella protothecoides can beengineered with a sucrose invertase gene under the regulatory control ofone of three promoters (Cauliflower mosaic virus 35S promoter (CMV),Chlorella virus promoter (CV), or Chlorella HUP1 promoter (HUP1)). Thesucrose invertase gene used in this example comprises a modification tothe S. cerevisiae SUC2 gene to optimize for C. protothecoides codonusage. The cDNA and amino acid sequences of the optimized genecorrespond to SEQ ID NO:8 and SEQ ID NO:19, respectively. Anillustration of the plasmid constructs used in the transformation isshown in FIG. 25. Expression of a secretable sucrose invertase, such asthat described herein, permits the use of molasses, sugar cane juice,and other sucrose-containing feedstocks for cell fermentation.

Similarly, FIGS. 27 and 28 show the results of transformation ofChlorella protothecoides, and Chlorella minutissima and Chlorellaemersonii, respectively, with the sucrose invertase gene from S.cerevisiae under the control of the CMV promoter.

The growth potential of microorganisms expressing an exogenoussecretable sucrose invertase is illustrated by the addition of aninvertase to the culture medium of Chlorella protothecoides, asdescribed in further detail in the Examples. FIGS. 23 and 24 illustratethe surprising result that Chlorella cells grow as well on wastemolasses from sugar cane processing as they do on pure reagent-gradeglucose; the use of this low-value waste product of sugar caneprocessing can provide significant cost savings in the production ofhydrocarbons and other oils. Molasses contains lignin and othercellulosic waste products that poison many microorganisms and retardtheir growth, however it was discovered that Chlorella cells thrive inthe presence of such poisons. FIGS. 23-24 show the growth of cells onthree unique sources of molasses (designated BS1, BS2 and HTM), ascompared to growth on glucose or sucrose in the presence or absence ofan extracellular sucrose invertase.

Alternatively, a sucrose invertase can also be expressed intracellularlyin cells that express a sucrose transporter, as well as in cells thatexpress any carbohydrate transporter that allows sucrose to enter thecell.

A foreign gene was transformed into and expressed in Chlorellaprotothecoides, as described in Example 12. Expression of sucroseutilization genes can be accomplished using the same or similarmethodology and vector design.

Bioreactors can be employed for use in heterotrophic growth methods. Aswill be appreciated, provisions made to make light available to thecells in photosynthetic growth methods are unnecessary when using afixed-carbon source in the heterotrophic growth methods describedherein.

The specific examples of process conditions and heterotrophic growthmethods described herein can be combined in any suitable manner toimprove efficiencies of microbial growth and lipid production. Inaddition, the invention includes the selection and/or geneticengineering of microbes, such as microalgae, to produce microbes thatare even more suitable for use in the above-described methods. Forexample, the microbes having a greater ability to utilize any of theabove-described feedstocks for increased proliferation and/or lipid(e.g., fatty acid) production are within the scope of the invention.

C. Mixotrophic Growth

Mixotrophic growth is the use of both light and fixed carbon source(s)as energy sources for cells to grow and produce hydrocarbons.Mixotrophic growth can be conducted in a photobioreactor. Microalgae canbe grown and maintained in closed photobioreactors made of differenttypes of transparent or semitransparent material. Such material caninclude Plexiglas® enclosures, glass enclosures, bags made fromsubstances such as polyethylene, transparent or semitransparent pipes,and other materials. Microalgae can be grown and maintained in openphotobioreactors such as raceway ponds, settling ponds, and othernon-enclosed containers.

D. Growth Media

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of lipid and/or hydrocarbon constituentscan be difficult to predict. In some cases, certain strains ofmicroorganisms may be unable to grow on a particular growth mediumbecause of the presence of some inhibitory component or the absence ofsome essential nutritional requirement required by the particular strainof microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin for its culturecollection of algae (UTEX). For example, various fresh water and saltwater media include those shown in Table 4, below.

TABLE 4 Exemplary Algal Media. Fresh Water Media Salt Water Media ½ CHEVDiatom Medium 1% F/2 ⅓ CHEV Diatom Medium ½ Enriched Seawater Medium ⅕CHEV Diatom Medium ½ Erdschreiber Medium 1:1 DYIII/PEA + Gr+ ½ Soil +Seawater Medium ⅔ CHEV Diatom Medium ⅓ Soil + Seawater Medium 2X CHEVDiatom Medium ¼ ERD Ag Diatom Medium ¼ Soil + Seawater Medium AllenMedium ⅕ Soil + Seawater Medium BG11-1 Medium ⅔ Enriched Sewater MediumBold 1NV Medium 20% Allen + 80% ERD Bold 3N Medium 2X Erdschreiber'sMedium Botryococcus Medium 2X Soil + Seawater Medium Bristol Medium 5%F/2 Medium CHEV Diatom Medium 5/3 Soil + Seawater Agar Medium Chu'sMedium Artificial Seawater Medium CR1 Diatom Medium BG11-1 + .36% NaClMedium CR1+ Diatom Medium BG11-1 + 1% NaCl Medium CR1-S Diatom MediumBold 1NV:Erdshreiber (1:1) Cyanidium Medium Bold 1NV:Erdshreiber (4:1)Cyanophycean Medium Bristol-NaCl Medium Desmid Medium DasycladalesSeawater Medium DYIII Medium Enriched Seawater Medium Euglena MediumErdschreiber's Medium HEPES Medium ES/10 Enriched Seawater Medium JMedium ES/2 Enriched Seawater Medium Malt Medium ES/4 Enriched SeawaterMedium MES Medium F/2 Medium Modified Bold 3N Medium F/2 + NH4 ModifiedCOMBO Medium LDM Medium N/20 Medium Modified 2 X CHEV Ochromonas MediumModified 2 X CHEV + Soil P49 Medium Modified Artificial Seawater MediumPolytomella Medium Modified CHEV Proteose Medium Porphridium Medium SnowAlgae Media Soil + Seawater Medium Soil Extract Medium SS Diatom MediumSoilwater: BAR Medium Soilwater: GR− Medium Soilwater: GR−/NH4 MediumSoilwater: GR+ Medium Soilwater: GR+/NH4 Medium Soilwater: PEA MediumSoilwater: Peat Medium Soilwater: VT Medium Spirulina Medium Tap MediumTrebouxia Medium Volvocacean Medium Volvocacean-3N Medium Volvox MediumVolvox-Dextrose Medium Waris Medium Waris + Soil Extract Medium

In a particular example, a medium suitable for culturing Chlorellaprotothecoides (UTEX 31) comprises Proteose Medium. This medium issuitable for axenic cultures, and a 1 L volume of the medium (pH ˜6.8)can be prepared by addition of 1 g of proteose peptone to 1 liter ofBristol Medium. Bristol medium comprises 2.94 mM NaNO₃, 0.17 mMCaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaClin an aqueous solution. For 1.5% agar medium, 15 g of agar can be addedto 1 L of the solution. The solution is covered and autoclaved, and thenstored at a refrigerated temperature prior to use.

Other suitable media for use with the methods of the invention can bereadily identified by consulting the URL identified above, or byconsulting other organizations that maintain cultures of microorganisms,such as SAG, CCAP, or CCALA. SAG refers to the Culture Collection ofAlgae at the University of Göttingen (Göttingen, Germany), CCAP refersto the culture collection of algae and protozoa managed by the ScottishAssociation for Marine Science (Scotland, United Kingdom), and CCALArefers to the culture collection of algal laboratory at the Institute ofBotany (T{hacek over (r)}ebo{hacek over (n)}, Czech Republic).

E. Increasing Yield of Lipids

Process conditions can be adjusted to increase the yield of lipidssuitable for a particular use and/or to reduce production cost. Forexample, in certain embodiments, a microbe (e.g., a microalgae) iscultured in the presence of a limiting concentration of one or morenutrients, such as, for example, carbon and/or nitrogen, phosphorous, orsulfur, while providing an excess of fixed carbon energy such asglucose. Nitrogen limitation tends to increase microbial lipid yieldover microbial lipid yield in a culture in which nitrogen is provided inexcess. In particular embodiments, the increase in lipid yield is atleast about: 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or500%. The microbe can be cultured in the presence of a limiting amountof a nutrient for a portion of the total culture period or for theentire period. In particular embodiments, the nutrient concentration iscycled between a limiting concentration and a non-limiting concentrationat least twice during the total culture period.

To increase lipid yield, acetic acid can be employed in the feedstockfor a lipid-producing microbe (e.g., a microalgae). Acetic acid feedsdirectly into the point of metabolism that initiates fatty acidsynthesis (i.e., acetyl-CoA); thus providing acetic acid in the culturecan increase fatty acid production. Generally, the microbe is culturedin the presence of a sufficient amount of acetic acid to increasemicrobial lipid yield, and/or microbial fatty acid yield, specifically,over microbial lipid (e.g., fatty acid) yield in the absence of aceticacid.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example:biotin, pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingconstructs and techniques such as those described above.

V. Lipid Pathway Engineering

In some embodiments of the present invention, microorganisms of thepresent invention are modified to alter the properties and/orproportions of lipids produced and/or to increase carbon flux intolipids. The pathway can further, or alternatively, be modified to alterthe properties and/or proportions of various hydrocarbon moleculesproduced through enzymatic processing of lipids.

A. Alteration of Properties or Proportions of Lipids or HydrocarbonsProduced

In the case of microalgae, some wild-type cells already have good growthcharacteristics but do not produce the desired types or quantities oflipids. Examples include Pyrobotrys, Phormidium, Agmenellum, Carteria,Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis, Anabaena, Euglena,Spirogyra, Chlorococcum, Tetraedron, Oscillatoria, Phagus, andChlorogonium, which have the desirable growth characteristic of growingin municipal sewage or wastewater. Such cells, as well as species ofChlorella and other microbes, can be engineered to have improved lipidproduction characteristics. Desired characteristics include optimizinglipid yield per unit volume and/or per unit time, carbon chain length(e.g., for biodiesel production or for industrial applications requiringhydrocarbon feedstock), reducing the number of double or triple bonds,optionally to zero, removing or eliminating rings and cyclic structures,and increasing the hydrogen:carbon ratio of a particular species oflipid or of a population of distinct lipid. In addition, microalgae thatproduce appropriate hydrocarbons can also be engineered to have evenmore desirable hydrocarbon outputs. Examples of such microalgae includespecies of the genus Chlorella.

1. Regulation of Enzymes that Control Branch Points in Fatty AcidSynthesis

In particular embodiments, one or more key enzymes that control branchpoints in metabolism to fatty acid synthesis can be up-regulated ordown-regulated to improve lipid production. Up-regulation can beachieved, for example, by transforming cells with expression constructsin which a gene encoding the enzyme of interest is expressed, e.g.,using a strong promoter and/or enhancer elements that increasetranscription. Such constructs can include a selectable marker such thatthe transformants can be subjected to selection, which can result inamplification of the construct and an increase in the expression levelof the encoded enzyme. Examples of enzymes suitable for up-regulationaccording to the methods of the invention include pyruvatedehydrogenase, which plays a role in converting pyruvate to acetyl-CoA(examples, some from microalgae, include Genbank accession numbersNP_(—)415392; AAA53047; Q1XDM1; and CAF05587). Up-regulation of pyruvatedehydrogenase can increase production of acetyl-CoA, and therebyincrease fatty acid synthesis. Acetyl-CoA carboxylase catalyzes theinitial step in fatty acid synthesis. Accordingly, this enzyme can beup-regulated to increase production of fatty acids (examples, some frommicroalgae, include Genbank accession numbers BAA94752; AAA75528;AAA81471; YP_(—)537052; YP_(—)536879; NP_(—)045833; and BAA57908). Fattyacid production can also be increased by up-regulation of acyl carrierprotein (ACP), which carries the growing acyl chains during fatty acidsynthesis (examples, some from microalgae, include Genbank accessionnumbers A0T0F8; P51280; NP_(—)849041; YP_(—)874433).Glycerol-3-phosphate acyltransferase catalyzes the rate-limiting step offatty acid synthesis. Up-regulation of this enzyme can increase fattyacid production (examples, some from microalgae, include Genbankaccession numbers AAA74319; AAA33122; AAA37647; P44857; and ABO94442).The preceding proteins are candidates for expression in microalge,including species of the genus Chlorella.

Down-regulation of an enzyme of interest can achieved using, e.g.,antisense, catalytic RNA/DNA, RNA interference (RNAi), “knock-out,”“knock-down,” or other mutagenesis techniques. Enzymeexpression/function can also be inhibited using intrabodies. Examples ofenzymes suitable for down-regulation according to the methods of theinvention include citrate synthase, which consumes acetyl-CoA as part ofthe tricarboxylic acid (TCA) cycle. Down-regulation of citrate synthasecan force more acetyl-CoA into the fatty acid synthetic pathway.

2. Modulation of Global Regulators of Fatty Acid Synthetic Genes

Global regulators modulate the expression of the genes of the fatty acidbiosynthetic pathways. Accordingly, one or more global regulators offatty acid synthesis can be up- or down-regulated, as appropriate, toinhibit or enhance, respectively, the expression of a plurality of fattyacid synthetic genes and, ultimately, to increase lipid production.Examples include sterol regulatory element binding proteins (SREBPs),such as SREBP-1a and SREBP-1c (for examples see Genbank accessionnumbers NP_(—)035610 and Q9WTN3). Global regulators can be up- ordown-regulated, for example, as described above with respect toregulation of control point enzymes.

3. Regulation of Hydrocarbon Modification Enzymes

The methods of the invention also include transforming cells with one ormore genes encoding hydrocarbon modification enzymes such as, forexample, a fatty acyl-ACP thioesterase (see examples in Table 5 withaccession numbers), a fatty acyl-CoA/aldehyde reductase (see examples inTable 6 with accession numbers), a fatty acyl-CoA reductase (seeexamples in Table 7 with accession numbers), a fatty aldehydedecarbonylase (see examples in Table 8 with accession numbers), a fattyaldehyde reductase, or a squalene synthase gene (see GenBank Accessionnumber AF205791). In some embodiments, genes encoding a fatty acyl-ACPthioesterase and a naturally co-expressed acyl carrier protein may betransformed into a cell, optionally with one or more genes encodingother hydrocarbon modification enzymes. In other embodiments, the ACPand the fatty acyl-ACP thioesterase may have an affinity for one anotherthat imparts an advantage when the two are used together in the microbesand methods of the present invention, irrespective of whether they areor are not naturally co-expressed in a particular tissue or organism.Thus, the present invention contemplates both naturally co-expressedpairs of these enzymes as well as those that share an affinity forinteracting with one another to facilitate cleavage of a length-specificcarbon chain from the ACP.

In still other embodiments, an exogenous gene encoding a desaturase canbe transformed into the cell in conjunction with one or more genesencoding other hydrocarbon modification enzymes in order to providemodifications with respect to hydrocarbon saturation. Stearoyl-ACPdesaturase (see, e.g., GenBank Accession numbers AAF15308; ABM45911; andAAY86086), for example, catalyzes the conversion of stearoyl-ACP tooleoyl-ACP. Up-regulation of this gene can increase the proportion ofmonounsaturated fatty acids produced by a cell; whereas down-regulationcan reduce the proportion of monounsaturates. Similarly, the expressionof one or more glycerolipid desaturases can be controlled to alter theratio of unsaturated to saturated fatty acids such as ω-6 fatty aciddesaturase, ω-3 fatty acid desaturase, or ω-6-oleate desaturase. In someembodiments, the desaturase can be selected with reference to a desiredcarbon chain length, such that the desaturase is capable of makinglocation specific modifications within a specified carbon-lengthsubstrate, or substrates having a carbon-length within a specifiedrange.

In particular embodiments, microbes of the present invention aregenetically engineered to express one or more exogenous genes selectedfrom a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase,a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehydedecarbonylase, or a naturally co-expressed acyl carrier protein.Suitable expression methods are described above with respect to theexpression of a lipase gene, including, among other methods, inducibleexpression and compartmentalized expression.

Without intending to be bound by any particular theory or cellularmechanism, a fatty acyl-ACP thioesterase cleaves a fatty acid from anacyl carrier protein (ACP) during lipid synthesis. Through furtherenzymatic processing, the cleaved fatty acid is then combined with acoenzyme to yield an acyl-CoA molecule. This acyl-CoA is the substratefor the enzymatic activity of a fatty acyl-CoA reductase to yield analdehyde, as well as for a fatty acyl-CoA/aldehyde reductase to yield analcohol. The aldehyde produced by the action of the fatty acyl-CoAreductase identified above is the substrate for further enzymaticactivity by either a fatty aldehyde reductase to yield an alcohol, or afatty aldehyde decarbonylase to yield an alkane or alkene.

The enzymes described directly above have a specificity for acting on asubstrate which includes a specific number of carbon atoms. For example,a fatty acyl-ACP thioesterase may have a specificity for cleaving afatty acid having 12 carbon atoms from the ACP. In some embodiments, theACP and the length-specific thioesterase may have an affinity for oneanother that makes them particularly useful as a combination (e.g., theexogenous ACP and thioesterase genes may be naturally co-expressed in aparticular tissue or organism from which they are derived). Therefore,in various embodiments, the microbe can contain an exogenous gene thatencodes a protein with specificity for catalyzing an enzymatic activity(e.g., cleavage of a fatty acid from an ACP, reduction of an acyl-CoA toan aldehyde or an alcohol, or conversion of an aldehyde to an alkane)with regard to the number of carbon atoms contained in the substrate.The enzymatic specificity can, in various embodiments, be for asubstrate having from 8 to 34 carbon atoms, preferably from 8 to 18carbon atoms, and more preferably from 10 to 14 carbon atoms. The mostpreferred specificity is for a substrate having 12 carbon atoms. Inother embodiments the specificity can be for 20 to 30 carbon atoms.

Fatty acyl-ACP thioesterases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 5.

TABLE 5 Fatty acyl-ACP thioesterases and GenBank accession numbers.Umbellularia californica fatty acyl-ACP thioesterase (GenBank #AAC49001)Cinnamomum camphora fatty acyl-ACP thioesterase (GenBank #Q39473)Umbellularia californica fatty acyl-ACP thioesterase (GenBank #Q41635)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71729)Myristica fragrans fatty acyl-ACP thioesterase (GenBank #AAB71730)Elaeis guineensis fatty acyl-ACP thioesterase (GenBank #ABD83939) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAD42220) Populustomentosa fatty acyl-ACP thioesterase (GenBank #ABC47311) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #NP_172327) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85387) Arabidopsisthaliana fatty acyl-ACP thioesterase (GenBank #CAA85388) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #Q9SQI3) Cuphea lanceolatafatty acyl-ACP thioesterase (GenBank #CAA54060) Cuphea hookeriana fattyacyl-ACP thioesterase (GenBank #AAC72882) Cuphea calophylla subsp.mesostemon fatty acyl-ACP thioesterase (GenBank #ABB71581) Cuphealanceolata fatty acyl-ACP thioesterase (GenBank #CAC19933) Elaeisguineensis fatty acyl-ACP thioesterase (GenBank #AAL15645) Cupheahookeriana fatty acyl-ACP thioesterase (GenBank #Q39513) Gossypiumhirsutum fatty acyl-ACP thioesterase (GenBank #AAD01982) Vitis viniferafatty acyl-ACP thioesterase (GenBank #CAN81819) Garcinia mangostanafatty acyl-ACP thioesterase (GenBank #AAB51525) Brassica juncea fattyacyl-ACP thioesterase (GenBank #ABI18986) Madhuca longifolia fattyacyl-ACP thioesterase (GenBank #AAX51637) Brassica napus fatty acyl-ACPthioesterase (GenBank #ABH11710) Oryza sativa (indica cultivar-group)fatty acyl-ACP thioesterase (GenBank #EAY86877) Oryza sativa (japonicacultivar-group) fatty acyl-ACP thioesterase (GenBank #NP_001068400)Oryza sativa (indica cultivar-group) fatty acyl-ACP thioesterase(GenBank #EAY99617) Cuphea hookeriana fatty acyl-ACP thioesterase(GenBank #AAC49269)

Fatty acyl-CoA/aldehyde reductases suitable for use with the microbesand methods of the invention include, without limitation, those listedin Table 6.

TABLE 6 Fatty acyl-CoA/aldehyde reductases listed by GenBank accessionnumbers. AAC45217, YP_047869, BAB85476, YP_001086217, YP_580344,YP_001280274, YP_264583, YP_436109, YP_959769, ZP_01736962, ZP_01900335,ZP_01892096, ZP_01103974, ZP_01915077, YP_924106, YP_130411,ZP_01222731, YP_550815, YP_983712, YP_001019688, YP_524762, YP_856798,ZP_01115500, YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156,YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936, AAR88762,ABK28586, NP_197634, CAD30694, NP_001063962, BAD46254, NP_001030809,EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537, NP_001052541,CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844, AAZ06658, CAA68190,CAA52019, and BAC84377

Fatty acyl-CoA reductases suitable for use with the microbes and methodsof the invention include, without limitation, those listed in Table 7.

TABLE 7 Fatty acyl-CoA reductases listed by GenBank accession numbers.NP_187805, ABO14927, NP_001049083, CAN83375, NP_191229, EAZ42242,EAZ06453, CAD30696, BAD31814, NP_190040, AAD38039, CAD30692, CAN81280,NP_197642, NP_190041, AAL15288, and NP_190042

Fatty aldehyde decarbonylases suitable for use with the microbes andmethods of the invention include, without limitation, those listed inTable 8.

TABLE 8 Fatty aldehyde decarbonylases listed by GenBank accessionnumbers. NP_850932, ABN07985, CAN60676, AAC23640, CAA65199, AAC24373,CAE03390, ABD28319, NP_181306, EAZ31322, CAN63491, EAY94825, EAY86731,CAL55686, XP_001420263, EAZ23849, NP_200588, NP_001063227, CAN83072,AAR90847, and AAR97643

Combinations of naturally co-expressed fatty acyl-ACP thioesterases andacyl carrier proteins are suitable for use with the microbes and methodsof the invention.

Additional examples of hydrocarbon modification enzymes include aminoacid sequences contained in, referenced in, or encoded by nucleic acidsequences contained or referenced in, any of the following U.S. Pat.Nos. 6,610,527; 6,451,576; 6,429,014; 6,342,380; 6,265,639; 6,194,185;6,114,160; 6,083,731; 6,043,072; 5,994,114; 5,891,697; 5,871,988;6,265,639, and further described in GenBank Accession numbers: AAO18435;ZP_(—)00513891; Q38710; AAK60613; AAK60610; AAK60611; NP_(—)113747;CAB75874; AAK60612; AAF20201; BAA11024; AF205791; and CAA03710.

Other suitable enzymes for use with the microbes and the methods of theinvention include those that have at least 70% amino acid identity withone of the proteins listed in Tables 5-8, and that exhibit thecorresponding desired enzymatic activity (e.g., cleavage of a fatty acidfrom an acyl carrier protein, reduction of an acyl-CoA to an aldehyde oran alcohol, or conversion of an aldehyde to an alkane). In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth.

The hydrocarbon modification enzymes described above are useful in theproduction of various hydrocarbons from a microbe (e.g., a microalgae,an oleaginous yeast, or a fungus) or population of microbes, whereby afatty acyl-ACP thioesterase cleaves a fatty acid from an acyl carrierprotein (ACP) during lipid synthesis. Through further enzymaticprocessing, the cleaved fatty acid is then combined with a coenzyme toyield an acyl-CoA molecule. This acyl-CoA is the substrate for theenzymatic activity of a fatty acyl-CoA reductase to yield an aldehyde,as well as for a fatty acyl-CoA/aldehyde reductase to yield an alcohol.The aldehyde produced by the action of the fatty acyl-CoA reductaseidentified above is the substrate for further enzymatic activity byeither a fatty aldehyde reductase to yield an alcohol, or a fattyaldehyde decarbonylase to yield an alkane or alkene.

The hydrocarbon modification enzymes have a specificity for acting on asubstrate which includes a specific number of carbon atoms. For example,a fatty acyl-ACP thioesterase may have a specificity for cleaving afatty acid having 12 carbon atoms from the ACP. Therefore, in variousembodiments, the microbe can contain an exogenous gene that encodes aprotein with specificity for catalyzing an enzymatic activity (e.g.,cleavage of a fatty acid from an ACP, reduction of an acyl-CoA to analdehyde or an alcohol, or conversion of an aldehyde to an alkane) withregard to the number of carbon atoms contained in the substrate. Theenzymatic specificity can, in various embodiments, be for a substratehaving from 8 to 34 carbon atoms, preferably from 8 to 18 carbon atoms,and more preferably from 10 to 14 carbon atoms. The most preferredspecificity is for a substrate having 12 carbon atoms. In otherembodiments the specificity can be for 20 to 30 carbon atoms.

In some embodiments, fatty acids, or the corresponding primary alcohols,aldehydes, alkanes or alkenes, generated by the methods describedherein, contain at least about 8, at least about 10, at least about 12,at least about 14, at least about 16, at least about 18, at least about20, at least about 22, at least about 24, at least about 26, at leastabout 28, at least about 30, at least about 32, or at least about 34carbon atoms or more. Preferred fatty acids for the production ofbiodiesel, renewable diesel, or jet fuel, or the corresponding primaryalcohols, aldehydes, alkanes and alkenes, for industrial applicationscontain at least about 8 carbon atoms or more. In certain embodiments,the above fatty acids, as well as the other corresponding hydrocarbonmolecules, are saturated (with no carbon-carbon double or triple bonds);mono unsaturated (single double bond); poly unsaturated (two or moredouble bonds); are linear (not cyclic); and/or have little or nobranching in their structures.

By selecting the desired combination of exogenous genes to be expressed,one can tailor the product generated by the microbe, which may then beextracted from the aqueous biomass. For example, the microbe cancontain: (i) an exogenous gene encoding a fatty acyl-ACP thioesterase;and, optionally, (ii) a naturally co-expressed acyl carrier protein oran acyl carrier protein otherwise having affinity for the fatty acyl-ACPthioesterase (or conversely); and, optionally, (iii) an exogenous geneencoding a fatty acyl-CoA/aldehyde reductase or a fatty acyl-CoAreductase; and, optionally, (iv) an exogenous gene encoding a fattyaldehyde reductase or a fatty aldehyde decarbonylase. The microbe, whencultured as described below, synthesizes a fatty acid linked to an ACPand the fatty acyl-ACP thioesterase catalyzes the cleavage of the fattyacid from the ACP to yield, through further enzymatic processing, afatty acyl-CoA molecule. When present, the fatty acyl-CoA/aldehydereductase catalyzes the reduction of the acyl-CoA to an alcohol.Similarly, the fatty acyl-CoA reductase, when present, catalyzes thereduction of the acyl-CoA to an aldehyde. In those embodiments in whichan exogenous gene encoding a fatty acyl-CoA reductase is present andexpressed to yield an aldehyde product, a fatty aldehyde reductase,encoded by the third exogenous gene, catalyzes the reduction of thealdehyde to an alcohol. Similarly, a fatty aldehyde decarbonylasecatalyzes the conversion of the aldehyde to an alkane or an alkene, whenpresent.

Genes encoding such enzymes can be obtained from cells already known toexhibit significant lipid production such as Chlorella protothecoides.Genes already known to have a role in lipid production, e.g., a geneencoding an enzyme that saturates double bonds, can be transformedindividually into recipient cells. However, to practice the invention itis not necessary to make a priori assumptions as to which genes arerequired. A library of DNA containing different genes, such as cDNAsfrom a good lipid-production organism, can be transformed into recipientcells. The cDNA is preferably in operable linkage with a promoter activein microalgae. Different recipient microalgae cells transformed by alibrary receive different genes from the library. Transformants havingimproved lipid production are identified though screening methods knownin the art, such as, for example, HPLC, gas chromatography, and massspectrometry methods of hydrocarbon analysis (for examples of suchanalysis, see Biomass and Bioenergy Vol. 6. No. 4. pp. 269-274 (1994);Experientia 38; 47-49 (1982); and Phytochemistry 65 (2004) 3159-3165).These transformants are then subjected to further transformation withthe original library and/or optionally interbred to generate a furtherround of organisms having improved lipid production. General proceduresfor evolving whole organisms to acquire a desired property are describedin, e.g., U.S. Pat. No. 6,716,631. Such methods entail, e.g.,introducing a library of DNA fragments into a plurality of cells,whereby at least one of the fragments undergoes recombination with asegment in the genome or an episome of the cells to produce modifiedcells. The modified cells are then screened for modified cells that haveevolved toward acquisition of the desired function. Vectors and methodsfor transformation are analogous to those discussed in connection withexpression of lipase genes.

Furthermore, subtractive libraries can be used to identify genes whosetranscription is induced under different conditions, especiallyconditions employed in culturing microorganisms for biodieselproduction, or for the production of hydrocarbons useful as a feedstockfor industrial applications. Subtractive libraries contain nucleotidesequences reflecting the differences between two different samples. Suchlibraries are prepared by procedures that include the steps ofdenaturing and hybridizing populations of polynucleotides (e.g., mRNA,cDNA, amplified sequences) from each sample. Sequences common to bothsamples hybridize and are removed, leaving the sequences that differbetween the samples. In this manner, sequences that are induced underparticular conditions can be identified. This technique can be employed,for example, to identify genes useful for increasing lipid (e.g., fattyacid) production and, in particular, lipid production under any desiredculture conditions. The subtractive hybridization technique can also beemployed to identify promoters, e.g., inducible promoters, useful inexpression constructs according to the invention.

Thus, for example, subtractive libraries can be prepared frommicroorganism cultures grown autotrophically (in the light without afixed carbon source) or heterotrophically (in the dark in the presenceof a fixed carbon source). In particular, heterotrophic genes may beinduced during dark growth in the presence of a fixed carbon source andmay therefore be present in a library generated by subtracting sequencesfrom autotrophic cells from sequences from dark heterotrophic cells.Subtractive libraries can also be prepared from cultures to which aparticular carbon substrate, such as glucose, has been added to identifygenes that play a role in metabolizing the substrate. Subtractivelibraries prepared from cultures grown in the presence of excess versuslimited nitrogen can be used to identify genes that control celldivision as opposed to hydrocarbon accumulation production. Thepreparation of a subtractive library from a culture to which lipids(e.g., fatty acids) have been added can help identify genes whoseoverexpression increases fatty acid production. More specifically, theaddition of fatty acids to a culture of cells that can use the addedfatty acids will lead to the down-regulation of fatty acid syntheticgenes to down-regulate fatty acid production. The overexpression of oneor more such genes will have the opposite effect.

B. Increased Carbon Flux into Lipid Pathway

Some microalgae produce significant quantities of non-lipid metabolites,such as, for example, polysaccharides. Because polysaccharidebiosynthesis can use a significant proportion of the total metabolicenergy available to cells, mutagenesis of lipid-producing cells followedby screening for reduced or eliminated polysaccharide productiongenerates novel strains that are capable of producing higher yields oflipids.

The phenol:sulfuric acid assay detects carbohydrates (see Hellebust,Handbook of Phycological Methods, Cambridge University Press, 1978; andCuesta G., et al., J Microbiol Methods. 2003 January; 52(1):69-73). The1,6 dimethylmethylene blue assay detects anionic polysaccharides. (seefor example Braz J Med Biol Res. 1999 May; 32(5):545-50; Clin Chem. 1986November; 32(11):2073-6).

Polysaccharides can also be analyzed through methods such as HPLC, sizeexclusion chromatography, and anion exchange chromatography (see forexample Prosky L, Asp N, Schweizer T F, DeVries J W & Furda I (1988)Determination of insoluble, soluble and total dietary fiber in food andfood products: Interlaboratory study. Journal of the Association ofOfficial Analytical Chemists 71, 1017±1023; Int J Biol Macromol. 2003November; 33(1-3):9-18). Polysaccharides can also be detected using gelelectrophoresis (see for example Anal Biochem. 2003 Oct. 15;321(2):174-82; Anal Biochem. 2002 Jan. 1; 300(1):53-68).

VI. Methods of Recovering Lipids and Hydrocarbons

Hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, andalkanes) produced by cells of the invention can be harvested, orotherwise collected, by any convenient means. For example, hydrocarbonssecreted from cells can be centrifuged to separate the hydrocarbons in ahydrophobic layer from contaminants in an aqueous layer and optionallyfrom any solid materials as a precipitate in after centrifugation.Material containing cell or cell fractions can be treated with proteasesto degrade contaminating proteins before or after centrifugation. Insome instances the contaminating proteins are associated, possiblycovalently, to hydrocarbons or hydrocarbon precursors which formhydrocarbons upon removal of the protein. In other instances thehydrocarbon molecules are in a preparation that also contains proteins.Proteases can be added to hydrocarbon preparations containing proteinsto degrade proteins (for example, the protease from Streptomyces griseuscan be used (SigmaAldrich catalog number P5147). After digestion, thehydrocarbons are preferably purified from residual proteins, peptidefragments, and amino acids. This purification can be accomplished, forexample, by methods listed above such as centrifugation and filtration.

Extracellular hydrocarbons can also be extracted in vivo from livingmicroalgae cells which are then returned to a bioreactor by exposure ofthe cells, in an otherwise sterile environment, to a non-toxicextraction solvent, followed by separation of the living cells and thehydrophobic fraction of extraction solvent and hydrocarbons, wherein theseparated living cells are then returned to a culture container such asa stainless steel fermentor or photobioreactor (see Biotechnol Bioeng.2004 Dec. 5; 88(5):593-600 and Biotechnol Bioeng. 2004 Mar. 5;85(5):475-81).

Hydrocarbons can also be isolated by whole cell extraction. The cellsare first disrupted, as described in the section entitled “LysingCells”, and then intracellular and cell membrane/cell wall-associatedhydrocarbons as well as extracellular hydrocarbons can be collected fromthe whole cell mass, such as by use of centrifugation as describedabove.

Various methods are available for separating hydrocarbons and lipidsfrom cellular lysates produced by the above methods. For example,hydrocarbons can be extracted with a hydrophobic solvent such as hexane(see Frenz et al. 1989, Enzyme Microb. Technol., 11:717). Hydrocarbonscan also be extracted using liquefaction (see for example Sawayama etal. 1999, Biomass and Bioenergy 17:33-39 and Inoue et al. 1993, BiomassBioenergy 6(4):269-274); oil liquefaction (see for example Minowa et al.1995, Fuel 74(12):1735-1738); and supercritical CO₂ extraction (see forexample Mendes et al. 2003, Inorganica Chimica Acta 356:328-334).

Miao and Wu describe a protocol of the recovery of microalgal lipid froma culture of Chlorella protothecoides in which the cells were harvestedby centrifugation, washed with distilled water and dried by freezedrying. The resulting cell powder was pulverized in a mortar and thenextracted with n-hexane. Miao and Wu, Biosource Technology (2006)97:841-846.

A. Lysing Cells

Intracellular lipids and hydrocarbons produced in microorganisms are, insome embodiments, extracted after lysing the cells of the microorganism.Once extracted, the lipids and/or hydrocarbons can be further refined toproduce oils, fuels, or oleochemicals.

After completion of culturing, the microorganisms can be separated fromthe fermentation broth. Optionally, the separation is effected bycentrifugation to generate a concentrated paste. Centrifugation does notremove significant amounts of intracellular water from themicroorganisms and is not a drying step. The biomass can then be washedwith a washing solution (e.g., DI water) to get rid of the fermentationbroth and debris. Optionally, the washed microbial biomass may also bedried (oven dried, lyophilized, etc.) prior to cell disruption.Alternatively, cells can be lysed without separation from some or all ofthe fermentation broth when the fermentation is complete. For example,the cells can be at a ratio of less than 1:1 v:v cells to extracellularliquid when the cells are lysed.

Microorganisms containing a lipid and/or hydrocarbon can be lysed toproduce a lysate. As detailed herein, the step of lysing a microorganism(also referred to as cell lysis) can be achieved by any convenientmeans, including heat-induced lysis, adding a base, adding an acid,using enzymes such as proteases and polysaccharide degradation enzymessuch as amylases, using ultrasound, mechanical lysis, using osmoticshock, infection with a lytic virus, and/or expression of one or morelytic genes. Lysis is performed to release intracellular molecules whichhave been produced by the microorganism. Each of these methods forlysing a microorganism can be used as a single method or in combinationsimultaneously or sequentially.

The extent of cell disruption can be observed by microscopic analysis.Using one or more of the methods described herein, typically more than70% cell breakage is observed. Preferably, cell breakage is more than80%, more preferably more than 90% and most preferred about 100%.

In particular embodiments, the microorganism is lysed after growth, forexample to increase the exposure of cellular lipid and/or hydrocarbonfor extraction or further processing. The timing of lipase expression(e.g., via an inducible promoter) or cell lysis can be adjusted tooptimize the yield of lipids and/or hydrocarbons. Below are described anumber of lysis techniques. These techniques can be used individually orin combination.

1. Heat-Induced Lysis

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises heating of a cellular suspension containing themicroorganism. In this embodiment, the fermentation broth containing themicroorganisms (or a suspension of microorganisms isolated from thefermentation broth) is heated until the microorganisms, i.e., the cellwalls and membranes of microorganisms degrade or breakdown. Typically,temperatures applied are at least 50° C. Higher temperatures, such as,at least 30° C. at least 60° C., at least 70° C., at least 80° C., atleast 90° C., at least 100° C., at least 110° C., at least 120° C., atleast 130° C. or higher are used for more efficient cell lysis.

Lysing cells by heat treatment can be performed by boiling themicroorganism. Alternatively, heat treatment (without boiling) can beperformed in an autoclave. The heat treated lysate may be cooled forfurther treatment.

Cell disruption can also be performed by steam treatment, i.e., throughaddition of pressurized steam. Steam treatment of microalgae for celldisruption is described, for example, in U.S. Pat. No. 6,750,048.

2. Lysis Using a Base

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding a base to a cellular suspensioncontaining the microorganism.

The base should be strong enough to hydrolyze at least a portion of theproteinaceous compounds of the microorganisms used. Bases which areuseful for solubilizing proteins are known in the art of chemistry.Exemplary bases which are useful in the methods of the present inventioninclude, but are not limited to, hydroxides, carbonates and bicarbonatesof lithium, sodium, potassium, calcium, and mixtures thereof. Apreferred base is KOH. Base treatment of microalgae for cell disruptionis described, for example, in U.S. Pat. No. 6,750,048.

3. Acidic Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises adding an acid to a cellular suspensioncontaining the microorganism. Acid lysis can be effected using an acidat a concentration of 10-500 mN or preferably 40-160 nM. Acid lysis ispreferably performed at above room temperature (e.g., at 40-160°, andpreferably a temperature of 50-130°. For moderate temperatures (e.g.,room temperature to 100° C. and particularly room temperature to 65°,acid treatment can usefully be combined with sonication or other celldisruption methods.

4. Lysing Cells Using Enzymes

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises lysing the microorganism by using anenzyme. Preferred enzymes for lysing a microorganism are proteases andpolysaccharide-degrading enzymes such as hemicellulase (e.g.,hemicellulase from Aspergillus niger; Sigma Aldrich, St. Louis, Mo.;#H2125), pectinase (e.g., pectinase from Rhizopus sp.; Sigma Aldrich,St. Louis, Mo.; #P2401), Mannaway 4.0 L (Novozymes), cellulase (e.g.,cellulose from Trichoderma viride; Sigma Aldrich, St. Louis, Mo.;#C9422), and driselase (e.g., driselase from Basidiomycetes sp.; SigmaAldrich, St. Louis, Mo.; #D9515.

a) Cellulases

In a preferred embodiment of the present invention, a cellulase forlysing a microorganism is a polysaccharide-degrading enzyme, optionallyfrom Chlorella or a Chlorella virus.

b) Proteases

Proteases such as Streptomyces griseus protease, chymotrypsin,proteinase K, proteases listed in Degradation of Polylactide byCommercial Proteases, Oda Y et al., Journal of Polymers and theEnvironment, Volume 8, Number 1, January 2000, pp. 29-32(4), and otherproteases can be used to lyse microorganisms. Other proteases that canbe used include Alcalase 2.4 FG (Novozymes) and Flavourzyme 100 L(Novozymes).

c) Combinations

Any combination of a protease and a polysaccharide-degrading enzyme canalso be used, including any combination of the preceding proteases andpolysaccharide-degrading enzymes.

5. Lysing Cells Using Ultrasound

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by using ultrasound, i.e.,sonication. Thus, cells can also by lysed with high frequency sound. Thesound can be produced electronically and transported through a metallictip to an appropriately concentrated cellular suspension. Thissonication (or ultrasonication) disrupts cellular integrity based on thecreation of cavities in cell suspension.

6. Mechanical Lysis

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by mechanical lysis. Cells can belysed mechanically and optionally homogenized to facilitate hydrocarbon(e.g., lipid) collection. For example, a pressure disrupter can be usedto pump a cell containing slurry through a restricted orifice valve.High pressure (up to 1500 bar) is applied, followed by an instantexpansion through an exiting nozzle. Cell disruption is accomplished bythree different mechanisms: impingement on the valve, high liquid shearin the orifice, and sudden pressure drop upon discharge, causing anexplosion of the cell. The method releases intracellular molecules.

Alternatively, a ball mill can be used. In a ball mill, cells areagitated in suspension with small abrasive particles, such as beads.Cells break because of shear forces, grinding between beads, andcollisions with beads. The beads disrupt the cells to release cellularcontents. Cells can also be disrupted by shear forces, such as with theuse of blending (such as with a high speed or Waring blender asexamples), the french press, or even centrifugation in case of weak cellwalls, to disrupt cells.

7. Lysing Cells by Osmotic Shock (Cytolysis)

In another preferred embodiment of the present invention, the step oflysing a microorganism is performed by applying an osmotic shock.

8. Infection with a Lytic Virus

In a preferred embodiment of the present invention, the step of lysing amicroorganism comprises infection of the microorganism with a lyticvirus. A wide variety of viruses are known to lyse microorganismssuitable for use in the present invention, and the selection and use ofa particular lytic virus for a particular microorganism is within thelevel of skill in the art.

For example, paramecium bursaria chlorella virus (PBCV-1) is theprototype of a group (family Phycodnaviridae, genus Chlorovirus) oflarge, icosahedral, plaque-forming, double-stranded DNA viruses thatreplicate in, and lyse, certain unicellular, eukaryotic chlorella-likegreen algae. Accordingly, any susceptible microalgae can be lysed byinfecting the culture with a suitable chlorella virus. Methods ofinfecting species of Chlorella with a chlorella virus are known. See forexample Adv. Virus Res. 2006; 66:293-336; Virology, 1999 Apr. 25;257(1):15-23; Virology, 2004 Jan. 5; 318(1):214-23; Nucleic Acids Symp.Ser. 2000; (44):161-2; J. Virol. 2006 March; 80(5):2437-44; and Annu.Rev. Microbiol. 1999; 53:447-94.

9. Autolysis (Expression of a Lytic Gene)

In another preferred embodiment of the present invention, the step oflysing a microorganism comprises autolysis. In this embodiment, amicroorganism according to the invention is genetically engineered toproduce a lytic protein that will lyse the microorganism. This lyticgene can be expressed using an inducible promoter so that the cells canfirst be grown to a desirable density in a fermentor, followed byinduction of the promoter to express the lytic gene to lyse the cells.In one embodiment, the lytic gene encodes a polysaccharide-degradingenzyme.

In certain other embodiments, the lytic gene is a gene from a lyticvirus. Thus, for example, a lytic gene from a Chlorella virus can beexpressed in an algal cell of the genus Chlorella, such as C.protothecoides.

Suitable expression methods are described herein with respect to theexpression of a lipase gene. Expression of lytic genes is preferablydone using an inducible promoter, such as a promoter active inmicroalgae that is induced by a stimulus such as the presence of a smallmolecule, light, heat, and other stimuli. Lytic genes from chlorellaviruses are known. For example, see Virology 260, 308-315 (1999); FEMSMicrobiology Letters 180 (1999) 45-53; Virology 263, 376-387 (1999); andVirology 230, 361-368 (1997).

B. Extraction of Lipids and Hydrocarbons

Lipids and hydrocarbons generated by the microorganisms of the presentinvention can be recovered by extraction with an organic solvent. Insome cases, the preferred organic solvent is hexane. Typically, theorganic solvent is added directly to the lysate without prior separationof the lysate components. In one embodiment, the lysate generated by oneor more of the methods described above is contacted with an organicsolvent for a period of time sufficient to allow the lipid and/orhydrocarbon components to form a solution with the organic solvent. Insome cases, the solution can then be further refined to recover specificdesired lipid or hydrocarbon components. Hexane extraction methods arewell known in the art.

VII. Methods of Processing Lipids and Hydrocarbons

A. Enzymatic Modification

Hydrocarbons (e.g., lipids, fatty acids, aldehydes, alcohols, andalkanes) produced by cells as described herein can be modified by theuse of one or more enzymes, including a lipase, as described above. Whenthe hydrocarbons are in the extracellular environment of the cells, theone or more enzymes can be added to that environment under conditions inwhich the enzyme modifies the hydrocarbon or completes its synthesisfrom a hydrocarbon precursor. Alternatively, the hydrocarbons can bepartially, or completely, isolated from the cellular material beforeaddition of one or more catalysts such as enzymes. Such catalysts areexogenously added, and their activity occurs outside the cell or invitro.

B. Thermal and Other Catalytic Modification

Hydrocarbons produced by cells in vivo, or enzymatically modified invitro, as described herein can be optionally further processed byconventional means. The processing can include “cracking” to reduce thesize, and thus increase the hydrogen:carbon ratio, of hydrocarbonmolecules. Catalytic and thermal cracking methods are routinely used inhydrocarbon and triglyceride oil processing. Catalytic methods involvethe use of a catalyst, such as a solid acid catalyst. The catalyst canbe silica-alumina or a zeolite, which result in the heterolytic, orasymmetric, breakage of a carbon-carbon bond to result in a carbocationand a hydride anion. These reactive intermediates then undergo eitherrearrangement or hydride transfer with another hydrocarbon. Thereactions can thus regenerate the intermediates to result in aself-propagating chain mechanism. Hydrocarbons can also be processed toreduce, optionally to zero, the number of carbon-carbon double, ortriple, bonds therein. Hydrocarbons can also be processed to remove oreliminate a ring or cyclic structure therein. Hydrocarbons can also beprocessed to increase the hydrogen:carbon ratio. This can include theaddition of hydrogen (“hydrogenation”) and/or the “cracking” ofhydrocarbons into smaller hydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking ofmicroalgae-produced hydrocarbons. In alternative embodiments, thefraction is treated with another catalyst, such as an organic compound,heat, and/or an inorganic compound. For processing of lipids intobiodiesel, a transesterification process is used as described in SectionIV herein.

Hydrocarbons produced via methods of the present invention are useful ina variety of industrial applications. For example, the production oflinear alkylbenzene sulfonate (LAS), an anionic surfactant used innearly all types of detergents and cleaning preparations, utilizeshydrocarbons generally comprising a chain of 10-14 carbon atoms. See,for example, U.S. Pat. Nos. 6,946,430; 5,506,201; 6,692,730; 6,268,517;6,020,509; 6,140,302; 5,080,848; and 5,567,359. Surfactants, such asLAS, can be used in the manufacture of personal care compositions anddetergents, such as those described in U.S. Pat. Nos. 5,942,479;6,086,903; 5,833,999; 6,468,955; and 6,407,044.

VIII. Methods of Producing Fuels Suitable for Use in Diesel Vehicles andJet Engines

Increasing interest is directed to the use of hydrocarbon components ofbiological origin in fuels, such as biodiesel, renewable diesel, and jetfuel, since renewable biological starting materials that may replacestarting materials derived from fossil fuels are available, and the usethereof is desirable. There is an urgent need for methods for producinghydrocarbon components from biological materials. The present inventionfulfills this need by providing methods for production of biodiesel,renewable diesel, and jet fuel using the lipids generated by the methodsdescribed herein as a biological material to produce biodiesel,renewable diesel, and jet fuel.

Traditional diesel fuels are petroleum distillates rich in paraffinichydrocarbons. They have boiling ranges as broad as 370° to 780° F.,which are suitable for combustion in a compression ignition engine, suchas a diesel engine vehicle. The American Society of Testing andMaterials (ASTM) establishes the grade of diesel according to theboiling range, along with allowable ranges of other fuel properties,such as cetane number, cloud point, flash point, viscosity, anilinepoint, sulfur content, water content, ash content, copper stripcorrosion, and carbon residue. Technically, any hydrocarbon distillatematerial derived from biomass or otherwise that meets the appropriateASTM specification can be defined as diesel fuel (ASTM D975), jet fuel(ASTM D1655), or as biodiesel (ASTM D6751).

After extraction, lipid and/or hydrocarbon components recovered from themicrobial biomass described herein can be subjected to chemicaltreatment to manufacture a fuel for use in diesel vehicles and jetengines.

A. Biodiesel

Biodiesel is a liquid which varies in color—between golden and darkbrown—depending on the production feedstock. It is practicallyimmiscible with water, has a high boiling point and low vapor pressure.Biodiesel refers to a diesel-equivalent processed fuel for use indiesel-engine vehicles. Biodiesel is biodegradable and non-toxic. Anadditional benefit of biodiesel over conventional diesel fuel is lowerengine wear.

Typically, biodiesel comprises C14-C18 alkyl esters. Various processesconvert biomass or a lipid produced and isolated as described herein todiesel fuels. A preferred method to produce biodiesel is bytransesterification of a lipid as described herein. A preferred alkylester for use as biodiesel is a methyl ester or ethyl ester.

Biodiesel produced by a method described herein can be used alone orblended with conventional diesel fuel at any concentration in mostmodern diesel-engine vehicles. When blended with conventional dieselfuel (petroleum diesel), biodiesel may be present from about 0.1% toabout 99.9%. Much of the world uses a system known as the “B” factor tostate the amount of biodiesel in any fuel mix. For example, fuelcontaining 20% biodiesel is labeled B20. Pure biodiesel is referred toas B100.

Biodiesel can also be used as a heating fuel in domestic and commercialboilers. Existing oil boilers may contain rubber parts and may requireconversion to run on biodiesel. The conversion process is usuallyrelatively simple, involving the exchange of rubber parts for syntheticparts due to biodiesel being a strong solvent. Due to its strong solventpower, burning biodiesel will increase the efficiency of boilers.

Biodiesel can be used as an additive in formulations of diesel toincrease the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel,which is advantageous because it has virtually no sulfur content.

Biodiesel is a better solvent than petrodiesel and can be used to breakdown deposits of residues in the fuel lines of vehicles that havepreviously been run on petrodiesel.

1. Production of Biodiesel

Biodiesel can be produced by transesterification of triglyceridescontained in oil-rich biomass. Thus, in another aspect of the presentinvention a method for producing biodiesel is provided. In a preferredembodiment, the method for producing biodiesel comprises the steps of(a) cultivating a lipid-containing microorganism using methods disclosedherein (b) lysing a lipid-containing microorganism to produce a lysate,(c) isolating lipid from the lysed microorganism, and (d)transesterifying the lipid composition, whereby biodiesel is produced.

Methods for growth of a microorganism, lysing a microorganism to producea lysate, treating the lysate in a medium comprising an organic solventto form a heterogeneous mixture and separating the treated lysate into alipid composition have been described above and can also be used in themethod of producing biodiesel.

Lipid compositions can be subjected to transesterification to yieldlong-chain fatty acid esters useful as biodiesel. Preferredtransesterification reactions are outlined below and include basecatalyzed transesterification and transesterification using recombinantlipases.

In a base-catalyzed transesterification process, the triacylglyceridesare reacted with an alcohol, such as methanol or ethanol, in thepresence of an alkaline catalyst, typically potassium hydroxide. Thisreaction forms methyl or ethyl esters and glycerin (glycerol) as abyproduct.

a). General Chemical Process

Animal and plant oils are typically made of triglycerides which areesters of free fatty acids with the trihydric alcohol, glycerol. Intransesterification, the glycerol in a triacylglyceride (TAG) isreplaced with a short-chain alcohol such as methanol or ethanol. Atypical reaction scheme is as follows:

In this scheme, the alcohol is deprotonated with a base to make it astronger nucleophile. Commonly, ethanol or methanol is used in vastexcess (up to 50-fold). Normally, this reaction will proceed eitherexceedingly slowly or not at all. Heat, as well as an acid or base canbe used to help the reaction proceed more quickly. The acid or base arenot consumed by the transesterification reaction, thus they are notreactants but catalysts. Almost all biodiesel has been produced usingthe base-catalyzed technique as it requires only low temperatures andpressures and produces over 98% conversion yield (provided the startingoil is low in moisture and free fatty acids).

b). Using Recombinant Lipases

Transesterification has also been carried out experimentally using anenzyme, such as a lipase instead of a base. Lipase-catalyzedtransesterification can be carried out, for example, at a temperaturebetween the room temperature and 80° C., and a mole ratio of the TAG tothe lower alcohol of greater than 1:1, preferably about 3:1.

Lipases suitable for use in transesterification include, but are notlimited to, those listed in Table 9. Other examples of lipases usefulfor transesterification are found in, e.g. U.S. Pat. Nos. 4,798,793;4,940,845 5,156,963; 5,342,768; 5,776,741 and WO89/01032.

TABLE 9 Lipases suitable for use in transesterification. Aspergillusniger lipase ABG73614, Candida antarctica lipase B (novozym-435)CAA83122, Candida cylindracea lipase AAR24090, Candida lipolytica lipase(Lipase L; Amano Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g.,Lipase-OF; Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM20), Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase(Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959, Rhizopusoryzae lipase (Lipase F) AAF32408, Serratia marcescens lipase (SMEnzyme) ABI13521, Thermomyces lanuginosa lipase CAB58509, Lipase P(Nagase ChemteX Corporation), and Lipase QLM (Meito Sangyo Co., Ltd.,Nagoya, Japan)

One challenge to using a lipase for the production of fatty acid esterssuitable for biodiesel is that the price of lipase is much higher thanthe price of sodium hydroxide (NaOH) used by the strong base process.This challenge has been addressed by using an immobilized lipase, whichcan be recycled. However, the activity of the immobilized lipase must bemaintained after being recycled for a minimum number of cycles to allowa lipase-based process to compete with the strong base process in termsof the production cost. Immobilized lipases are subject to poisoning bythe lower alcohols typically used in transesterification. U.S. Pat. No.6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods forenhancing the activity of immobilized lipases and regeneratingimmobilized lipases having reduced activity.

In particular embodiments, a recombinant lipase is expressed in the samemicroorganisms that produce the lipid on which the lipase acts. Suitablerecombinant lipases include those listed above in Table 9 and/or havingGenBank Accession numbers listed above in Table 9, or a polypeptide thathas at least 70% amino acid identity with one of the lipases listedabove in Table 9 and that exhibits lipase activity. In additionalembodiments, the enzymatic activity is present in a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% identity with one of theabove described sequences, all of which are hereby incorporated byreference as if fully set forth. DNA encoding the lipase and selectablemarker is preferably codon-optimized cDNA. Methods of recoding genes forexpression in microalgae are described in U.S. Pat. No. 7,135,290.

2. Standards

The common international standard for biodiesel is EN 14214. ASTM D6751is the most common biodiesel standard referenced in the United Statesand Canada. Germany uses DIN EN 14214 and the UK requires compliancewith BS EN 14214.

Basic industrial tests to determine whether the products conform tothese standards typically include gas chromatography, HPLC, and others.Biodiesel meeting the quality standards is very non-toxic, with atoxicity rating (LD₅₀) of greater than 50 mL/kg.

B. Renewable Diesel

Renewable diesel comprises alkanes, such as C16:0 and C18:0 and thus,are distinguishable from biodiesel. High quality renewable dieselconforms to the ASTM D975 standard.

The lipids produced by the methods of the present invention can serve asfeedstock to produce renewable diesel. Thus, in another aspect of thepresent invention, a method for producing renewable diesel is provided.Renewable diesel can be produced by at least three processes:hydrothermal processing (hydrotreating); hydroprocessing; and indirectliquefaction. These processes yield non-ester distillates. During theseprocesses, triacylglycerides produced and isolated as described herein,are converted to alkanes.

In a preferred embodiment, the method for producing renewable dieselcomprises (a) cultivating a lipid-containing microorganism using methodsdisclosed herein (b) lysing the microorganism to produce a lysate, (c)isolating lipid from the lysed microorganism, and (d) deoxygenating andhydrotreating the lipid to produce an alkane, whereby renewable dieselis produced. Lipids suitable for manufacturing renewable diesel can beobtained via extraction from microbial biomass using an organic solventsuch as hexane, or via other methods, such as those described in U.S.Pat. No. 5,928,696.

In some methods, the microbial lipid is first cracked in conjunctionwith hydrotreating to reduce carbon chain length and saturate doublebonds, respectively. The material is then isomerized, also inconjunction with hydrotreating. The naptha fraction can then be removedthrough distillation, followed by additional distillation to vaporizeand distill components desired in the diesel fuel to meet a D975standard while leaving components that are heavier than desired formeeting a D 975 standard. Hydrotreating, hydrocracking, deoxygenationand isomerization methods of chemically modifying oils, includingtriglyceride oils, are well known in the art. See for example Europeanpatent applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;5,885,440; 6,881,873.

1. Hydrotreating

In a preferred embodiment of the method for producing renewable diesel,treating the lipid to produce an alkane is performed by hydrotreating ofthe lipid composition. In hydrothermal processing, typically, biomass isreacted in water at an elevated temperature and pressure to form oilsand residual solids. Conversion temperatures are typically 300° to 660°F., with pressure sufficient to keep the water primarily as a liquid,100 to 170 standard atmosphere (atm). Reaction times are on the order of15 to 30 minutes. After the reaction is completed, the organics areseparated from the water. Thereby a distillate suitable for diesel isproduced.

2. Hydroprocessing

A renewable diesel, referred to as “green diesel,” can be produced fromfatty acids by traditional hydroprocessing technology. Thetriglyceride-containing oils can be hydroprocessed either as co-feedwith petroleum or as a dedicated feed. The product is a diesel fuel thatconforms with the ASTM D975 specification. Thus, in another preferredembodiment of the method for producing renewable diesel, treating thelipid composition to produce an alkane is performed by hydroprocessingof the lipid composition.

In some methods of making renewable diesel, the first step of treating atriglyceride is hydroprocessing to saturate double bonds, followed bydeoxygenation at elevated temperature in the presence of hydrogen and acatalyst. In some methods, hydrogenation and deoxygenation occur in thesame reaction. In other methods deoxygenation occurs beforehydrogenation. Isomerization is then optionally performed, also in thepresence of hydrogen and a catalyst. Naphtha components are preferablyremoved through distillation. For examples, see U.S. Pat. No. 5,475,160(hydrogenation of triglycerides); U.S. Pat. No. 5,091,116(deoxygenation, hydrogenation and gas removal); U.S. Pat. No. 6,391,815(hydrogenation); and U.S. Pat. No. 5,888,947 (isomerization).

Petroleum refiners use hydroprocessing to remove impurities by treatingfeeds with hydrogen. Hydroprocessing conversion temperatures aretypically 300° to 700° F. Pressures are typically 40 to 100 atm. Thereaction times are typically on the order of 10 to 60 minutes.

Solid catalysts are employed to increase certain reaction rates, improveselectivity for certain products, and optimize hydrogen consumption.

Hydrotreating and hydroprocessing ultimately lead to a reduction in themolecular weight of the feed. In the case of triglyceride-containingoils, the triglyceride molecule is reduced to four hydrocarbon moleculesunder hydroprocessing conditions: a propane molecule and three heavierhydrocarbon molecules, typically in the C8 to C18 range.

3. Indirect Liquefaction

A traditional ultra-low sulfur diesel can be produced from any form ofbiomass by a two-step process. First, the biomass is converted to asyngas, a gaseous mixture rich in hydrogen and carbon monoxide. Then,the syngas is catalytically converted to liquids. Typically, theproduction of liquids is accomplished using Fischer-Tropsch (FT)synthesis. This technology applies to coal, natural gas, and heavy oils.Thus, in yet another preferred embodiment of the method for producingrenewable diesel, treating the lipid composition to produce an alkane isperformed by indirect liquefaction of the lipid composition.

C. Jet Fuel

The annual U.S. usage of jet fuel in 2006 was about 21 billion gallons(about 80 billion liters). Aeroplane fuel is clear to straw colored. Themost common fuel is an unleaded/paraffin oil-based fuel classified asAeroplane A-1, which is produced to an internationally standardized setof specifications. Aeroplane fuel is a mixture of a large number ofdifferent hydrocarbons, possibly as many as a thousand or more. Therange of their sizes (molecular weights or carbon numbers) is restrictedby the requirements for the product, for example, freezing point orsmoke point. Kerosone-type Aeroplane fuel (including Jet A and Jet A-1)has a carbon number distribution between about 8 and 16 carbon numbers.Wide-cut or naphta-type Aeroplane fuel (including Jet B) typically has acarbon number distribution between about 5 and 15 carbons.

Both Aeroplanes (Jet A and jet B) may contain a number of additives.Useful additives include, but are not limited to, antioxidants,antistatic agents, corrosion inhibitors, and fuel system icing inhibitor(FSII) agents. Antioxidants prevent gumming and usually, are based onalkylated phenols, for example, AO-30, AO-31, or AO-37. Antistaticagents dissipate static electricity and prevent sparking. Stadis 450with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, isan example. Corrosion inhibitors, e.g., DCI-4A is used for civilian andmilitary fuels and DCI-6A is used for military fuels. FSII agents,include, e.g., Di-EGME.

A solution is blending algae fuels with existing jet fuel. The presentinvention provides such a solution. The lipids produced by the methodsof the present invention can serve as feedstock to produce jet fuel.Thus, in another aspect of the present invention, a method for producingjet fuel is provided. Herewith two methods for producing jet fuel fromthe lipids produced by the methods of the present invention areprovided: fluid catalytic cracking (FCC); and hydrodeoxygenation (HDO).

1. Fluid Catalytic Cracking

Fluid Catalytic Cracking (FCC) is one method which is used to produceolefins, especially propylene from heavy crude fractions. There arereports in the literature that vegetable oils such as canola oil couldbe processed using FCC to give a hydrocarbon stream useful as a gasolinefuel.

The lipids produced by the method of the present invention can beconverted to olefins. The process involves flowing the lipids producedthrough an FCC zone and collecting a product stream comprised ofolefins, which is useful as a jet fuel. The lipids produced arecontacted with a cracking catalyst at cracking conditions to provide aproduct stream comprising olefins and hydrocarbons useful as jet fuel.

In a preferred embodiment, the method for producing jet fuel comprises(a) cultivating a lipid-containing microorganism using methods disclosedherein, (b) lysing the lipid-containing microorganism to produce alysate, (c) isolating lipid from the lysate, and (d) treating the lipidcomposition, whereby jet fuel is produced.

In a preferred embodiment of the method for producing a jet fuel, thelipid composition can be flowed through a fluid catalytic cracking zone,which, in one embodiment, may comprise contacting the lipid compositionwith a cracking catalyst at cracking conditions to provide a productstream comprising C₂-C₅ olefins.

In certain embodiments of this method it may be desirable to remove anycontaminants that may be present in the lipid composition. Thus, priorto flowing the lipid composition through a fluid catalytic crackingzone, the lipid composition is pretreated. Pretreatment may involvecontacting the lipid composition with an ion-exchange resin. The ionexchange resin is an acidic ion exchange resin, such as Amberlyst™-15and can be used as a bed in a reactor through which the lipidcomposition is flowed, either upflow or downflow. Other pretreatmentsmay include mild acid washes by contacting the lipid composition with anacid, such as sulfuric, acetic, nitric, or hydrochloric acid. Contactingis done with a dilute acid solution usually at ambient temperature andatmospheric pressure.

The lipid composition, optionally pretreated, is flowed to an FCC zonewhere the hydrocarbonaceous components are cracked to olefins. Catalyticcracking is accomplished by contacting the lipid composition in areaction zone with a catalyst composed of finely divided particulatematerial. The reaction is catalytic cracking, as opposed tohydrocracking, and is carried out in the absence of added hydrogen orthe consumption of hydrogen. As the cracking reaction proceeds,substantial amounts of coke are deposited on the catalyst. The catalystis regenerated at high temperatures by burning coke from the catalyst ina regeneration zone. Coke-containing catalyst, referred to herein as“coked catalyst”, is continually transported from the reaction zone tothe regeneration zone to be regenerated and replaced by essentiallycoke-free regenerated catalyst from the regeneration zone. Fluidizationof the catalyst particles by various gaseous streams allows thetransport of catalyst between the reaction zone and regeneration zone.Methods for cracking hydrocarbons, such as those of the lipidcomposition described herein, in a fluidized stream of catalyst,transporting catalyst between reaction and regeneration zones, andcombusting coke in the regenerator are well known by those skilled inthe art of FCC processes. Exemplary FCC applications and catalystsuseful for cracking the lipid composition to produce C₂-C₅ olefins aredescribed in U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporatedin their entirety by reference.

In one embodiment, cracking the lipid composition of the presentinvention, takes place in the riser section or, alternatively, the liftsection, of the FCC zone. The lipid composition is introduced into theriser by a nozzle resulting in the rapid vaporization of the lipidcomposition. Before contacting the catalyst, the lipid composition willordinarily have a temperature of about 149° C. to about 316° C. (300° F.to 600° F.). The catalyst is flowed from a blending vessel to the riserwhere it contacts the lipid composition for a time of abort 2 seconds orless.

The blended catalyst and reacted lipid composition vapors are thendischarged from the top of the riser through an outlet and separatedinto a cracked product vapor stream including olefins and a collectionof catalyst particles covered with substantial quantities of coke andgenerally referred to as “coked catalyst.” In an effort to minimize thecontact time of the lipid composition and the catalyst which may promotefurther conversion of desired products to undesirable other products,any arrangement of separators such as a swirl arm arrangement can beused to remove coked catalyst from the product stream quickly. Theseparator, e.g. swirl arm separator, is located in an upper portion of achamber with a stripping zone situated in the lower portion of thechamber. Catalyst separated by the swirl arm arrangement drops down intothe stripping zone. The cracked product vapor stream comprising crackedhydrocarbons including light olefins and some catalyst exit the chambervia a conduit which is in communication with cyclones. The cyclonesremove remaining catalyst particles from the product vapor stream toreduce particle concentrations to very low levels. The product vaporstream then exits the top of the separating vessel. Catalyst separatedby the cyclones is returned to the separating vessel and then to thestripping zone. The stripping zone removes adsorbed hydrocarbons fromthe surface of the catalyst by counter-current contact with steam.

Low hydrocarbon partial pressure operates to favor the production oflight olefins. Accordingly, the riser pressure is set at about 172 to241 kPa (25 to 35 psia) with a hydrocarbon partial pressure of about 35to 172 kPa (5 to 25 psia), with a preferred hydrocarbon partial pressureof about 69 to 138 kPa (10 to 20 psia). This relatively low partialpressure for hydrocarbon is achieved by using steam as a diluent to theextent that the diluent is 10 to 55 wt-% of lipid composition andpreferably about 15 wt-% of lipid composition. Other diluents such asdry gas can be used to reach equivalent hydrocarbon partial pressures.

The temperature of the cracked stream at the riser outlet will be about510° C. to 621° C. (950° F. to 1150° F.). However, riser outlettemperatures above 566° C. (1050° F.) make more dry gas and moreolefins. Whereas, riser outlet temperatures below 566° C. (1050° F.)make less ethylene and propylene. Accordingly, it is preferred to runthe FCC process at a preferred temperature of about 566° C. to about630° C., preferred pressure of about 138 kPa to about 240 kPa (20 to 35psia). Another condition for the process is the catalyst to lipidcomposition ratio which can vary from about 5 to about 20 and preferablyfrom about 10 to about 15.

In one embodiment of the method for producing a jet fuel, the lipidcomposition is introduced into the lift section of an FCC reactor. Thetemperature in the lift section will be very hot and range from about700° C. (1292° F.) to about 760° C. (1400° F.) with a catalyst to lipidcomposition ratio of about 100 to about 150. It is anticipated thatintroducing the lipid composition into the lift section will produceconsiderable amounts of propylene and ethylene.

Gas and liquid hydrocarbon products produced can be analyzed by gaschromatography, HPLC, etc.

2. Hydrodeoxygenation

In another embodiment of the method for producing a jet fuel using thelipid composition or the lipids produced as described herein, thestructure of the lipid composition or the lipids is broken by a processreferred to as hydrodeoxygenation (HDO).

HDO means removal of oxygen by means of hydrogen, that is, oxygen isremoved while breaking the structure of the material. Olefinic doublebonds are hydrogenated and any sulphur and nitrogen compounds areremoved. Sulphur removal is called hydrodesulphurization (HDS).Pretreatment and purity of the raw materials (lipid composition or thelipids) contribute to the service life of the catalyst.

Generally in the HDO/HDS step, hydrogen is mixed with the feed stock(lipid composition or the lipids) and then the mixture is passed througha catalyst bed as a co-current flow, either as a single phase or a twophase feed stock. After the HDO/MDS step, the product fraction isseparated and passed to a separate isomerization reactor. Anisomerization reactor for biological starting material is described inthe literature (FI 100 248) as a co-current reactor.

The process for producing a fuel by hydrogenating a hydrocarbon feed,e.g., the lipid composition or the lipids herein, can also be performedby passing the lipid composition or the lipids as a co-current flow withhydrogen gas through a first hydrogenation zone, and thereafter thehydrocarbon effluent is further hydrogenated in a second hydrogenationzone by passing hydrogen gas to the second hydrogenation zone as acounter-current flow relative to the hydrocarbon effluent. Exemplary HDOapplications and catalysts useful for cracking the lipid composition toproduce C₂-C₅ olefins are described in U.S. Pat. No. 7,232,935, which isincorporated in its entirety by reference.

Typically, in the hydrodeoxygenation step, the structure of thebiological component, such as the lipid composition or lipids herein, isdecomposed, oxygen, nitrogen, phosphorus and sulphur compounds, andlight hydrocarbons as gas are removed, and the olefinic bonds arehydrogenated. In the second step of the process, i.e. in the so-calledisomerization step, isomerization is carried out for branching thehydrocarbon chain and improving the performance of the paraffin at lowtemperatures.

In the first step i.e. HDO step of the cracking process, hydrogen gasand the lipid composition or lipids herein which are to be hydrogenatedare passed to a HDO catalyst bed system either as co-current orcounter-current flows, said catalyst bed system comprising one or morecatalyst bed(s), preferably 1-3 catalyst beds. The HDO step is typicallyoperated in a co-current manner. In case of a HDO catalyst bed systemcomprising two or more catalyst beds, one or more of the beds may beoperated using the counter-current flow principle.

In the HDO step, the pressure varies between 20 and 150 bar, preferablybetween 50 and 100 bar, and the temperature varies between 200 and 500°C., preferably in the range of 300-400° C.

In the HDO step, known hydrogenation catalysts containing metals fromGroup VII and/or VIB of the Periodic System may be used. Preferably, thehydrogenation catalysts are supported Pd, Pt, Ni, NiMo or a CoMocatalysts, the support being alumina and/or silica. Typically,NiMo/Al₂O₃ and CoMo/Al₂O₃ catalysts are used.

Prior to the HDO step, the lipid composition or lipids herein mayoptionally be treated by prehydrogenation under milder conditions thusavoiding side reactions of the double bonds. Such prehydrogenation iscarried out in the presence of a prehydrogenation catalyst attemperatures of 50 400° C. and at hydrogen pressures of 1 200 bar,preferably at a temperature between 150 and 250° C. and at a hydrogenpressure between 10 and 100 bar. The catalyst may contain metals fromGroup VIII and/or VIB of the Periodic System. Preferably, theprehydrogenation catalyst is a supported Pd, Pt, Ni, NiMo or a CoMocatalyst, the support being alumina and/or silica.

A gaseous stream from the HDO step containing hydrogen is cooled andthen carbon monoxide, carbon dioxide, nitrogen, phosphorus and sulphurcompounds, gaseous light hydrocarbons and other impurities are removedtherefrom. After compressing, the purified hydrogen or recycled hydrogenis returned back to the first catalyst bed and/or between the catalystbeds to make up for the withdrawn gas stream. Water is removed from thecondensed liquid. The liquid is passed to the first catalyst bed orbetween the catalyst beds.

After the HDO step, the product is subjected to an isomerization step.It is substantial for the process that the impurities are removed ascompletely as possible before the hydrocarbons are contacted with theisomerization catalyst. The isomerization step comprises an optionalstripping step, wherein the reaction product from the HDO step may bepurified by stripping with water vapour or a suitable gas such as lighthydrocarbon, nitrogen or hydrogen. The optional stripping step iscarried out in counter-current manner in a unit upstream of theisomerization catalyst, wherein the gas and liquid are contacted witheach other, or before the actual isomerization reactor in a separatestripping unit utilizing counter-current principle.

After the stripping step the hydrogen gas and the hydrogenated lipidcomposition or lipids herein, and optionally an n-paraffin mixture, arepassed to a reactive isomerization unit comprising one or severalcatalyst bed(s). The catalyst beds of the isomerization step may operateeither in co-current or counter-current manner.

It is important for the process that the counter-current flow principleis applied in the isomerization step. In the isomerization step this isdone by carrying out either the optional stripping step or theisomerization reaction step or both in counter-current manner.

The isomerization step and the HDO step may be carried out in the samepressure vessel or in separate pressure vessels. Optionalprehydrogenation may be carried out in a separate pressure vessel or inthe same pressure vessel as the HDO and isomerization steps.

In the isomerization step, the pressure varies in the range of 20 150bar, preferably in the range of 20 100 bar, the temperature beingbetween 200 and 500° C., preferably between 300 and 400° C.

In the isomerization step, isomerization catalysts known in the art maybe used. Suitable isomerization catalysts contain molecular sieve and/ora metal from Group VII and/or a carrier. Preferably, the isomerizationcatalyst contains SAPO-11 or SAPO41 or ZSM-22 or ZSM-23 or ferrieriteand Pt, Pd or N1 and Al₂O₃ or SiO₂. Typical isomerization catalysts are,for example, Pt/SAPO-11/Al₂O₃, Pt/ZSM-22/Al₂O₃, Pt/ZSM-23/Al₂O₃ andPt/SAPO-11/SiO₂.

As the product, a high quality hydrocarbon component of biologicalorigin, useful as a diesel fuel or a component thereof, is obtained, thedensity, cetane number and performance at low temperate of saidhydrocarbon component being excellent.

IX. Microbe Engineering

As noted above, in certain embodiments of the present invention it isdesirable to genetically modify a microorganism to enhance lipidproduction, modify the properties or proportions of components generatedby the microorganism, or to improve or provide de novo growthcharacteristics on a variety of feedstock materials. Chlorella,particularly Chlorella protothecoides, Chlorella minutissima, Chlorellasorokiniana, Chlorella ellipsoidea, Chlorella sp., and Chlorellaemersonii are preferred microorganisms for use in the geneticengineering methods described herein, although other Chlorella speciesas well as other varieties of microorganisms can be used.

Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (see for example Molecular Cloning: A LaboratoryManual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; andU.S. Pat. No. 4,683,202). Alternatively, elements can be generatedsynthetically using known methods (see for example Gene. 1995 Oct. 16;164(1):49-53).

A. Codon-Optimization for Expression

DNA encoding a polypeptide to be expressed in a microorganism, e.g., alipase and selectable marker are preferably codon-optimized cDNA.Methods of recoding genes for expression in microalgae are described inU.S. Pat. No. 7,135,290. Additional information for codon optimizationis available, e.g., at the codon usage database of GenBank. Asnon-limiting examples, codon usage in Chlorella pyrenoidosa, Dunaliellasalina, and Chlorella protothecoides are shown in Tables 10, 11, and 12,respectively.

TABLE 10 Codon usage in Chlorella pyrenoidosa. Phe UUU  39 (0.82) SerUCU 50 (1.04) UUC  56 (1.18) UCC 60 (1.25) Leu UUA  10 (0.20) UCA  6(0.96) UUG  46 (0.91) UCG 43 (0.89) Tyr UAU  15 (0.59) Cys UGU 46 (0.77)UAC  36 (1.41) UGC 73 (1.23) ter UAA  9 (0.00) ter UGA 43 (0.00) ter UAG 15 (0.00) Trp UGG 69 (1.00) Leu CUU  49 (0.97) Pro CCU 80 (0.98) CUC 73 (1.45) CCC 88 (1.08) CUA  22 (0.44) CCA 93 (1.14) CUG 103 (2.04) CCG65 (0.80) His CAU  50 (0.88) Arg CGU 39 (0.76) CAC  3 (1.12) CGC 63(1.23) Gln CAA  59 (0.84) CGA 46 (0.90) CAG  2 (1.16) CGG 47 (0.92) IleAUU  24 (0.69) Thr ACU 32 (0.67) AUC  61 (1.76) ACC 76 (1.60) AUA  19(0.55) ACA 41 (0.86) Met AUG  42 (1.00) ACG 41 (0.86) Asn AAU  26 (0.75)Ser AGU 23 (0.48) AAC  3 (1.25) AGC 67 (1.39) Lys AAA  32 (0.54) Arg AGA51 (1.00) AAG  86 (1.46) AGG 61 (1.19) Val GUU  36 (0.75) Ala GCU 57(0.79) GUC  54 (1.13) GCC 97 (1.34) GUA  30 (0.63) GCA 89 (1.23) GUG  71(1.49) GCG 47 (0.65) Asp GAU  60 (0.95) Gly GGU 35 (0.60) GAC  66 (1.05)GGC 78 (1.33) Glu GAA  41 (0.68) GGA 54 (0.92) GAG  80 (1.32) GGG 67(1.15)

TABLE 11 Preferred codon usage in Dunaliella salina. TTC (Phe) TAC (Tyr)TGC (Cys) TAA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg) CTG (Leu)CAG (Gln) ATC (Ile) ACC (Thr) AAC (Asn) AGC (Ser) ATG (Met) AAG (Lys)GCC (Ala) GAC (Asp) GGC (Gly) GTG (Val) GAG (Glu)

TABLE 12 Preferred codon usage in Chlorella protothecoides. TTC (Phe)TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp) CCC (Pro) CAC (His) CGC (Arg)CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) GAC (Asp) TCC (Ser) ATG (Met)AAG (Lys) GCC (Ala) AAC (Asn) GGC (Gly) GTG (Val) GAG (Glu)

B. Promoters

Many promoters are active in microalgae, including promoters that areendogenous to the algae being transformed, as well as promoters that arenot endogenous to the algae being transformed (i.e., promoters fromother algae, promoters from higher plants, and promoters from plantviruses or algae viruses). Exogenous and/or endogenous promoters thatare active in microalgae, and antibiotic resistance genes functional inmicroalgae are described by e.g., Curr Microbiol. 1997 December;35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). 2002 January;4(1):63-73 (Chlorella ellipsoidea); Mol Gen Genet. 1996 Oct. 16;252(5):572-9 (Phaeodactylum tricornutum); Plant Mol Biol. 1996 April;31(1):1-12 (Volvox carteri); Proc Natl Acad Sci USA. 1994 Nov. 22;91(24):11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc C,Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251 (Laboratoryof Molecular Plant Biology, Stazione Zoologica, Villa Comunale, I-80121Naples, Italy) (Phaeodactylum tricornutum and Thalassiosiraweissflogii); Plant Physiol. 2002 May; 129(1):7-12. (Porphyridium sp.);Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42. (Chlamydomonasreinhardtii); Proc Natl Acad Sci USA. 1990 February; 87(3):1228-32.(Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun. 25;20(12):2959-65; Mar Biotechnol (NY). 2002 January; 4(1):63-73(Chlorella); Biochem Mol Biol Int. 1995 August; 36(5):1025-35(Chlamydomonas reinhardtii); J. Microbiol. 2005 August; 43(4):361-5(Dunaliella); Yi Chuan Xue Bao. 2005 April; 32(4):424-33 (Dunaliella);Mar Biotechnol (NY). 1999 May; 1(3):239-251. (Thalassiosira andPhaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 February;58(2):123-37 (various species); Mol Genet Genomics. 2004 February;271(1):50-9 (Thermosynechococcus elongates); J. Bacteriol. (2000), 182,211-215; FEMS Microbiol Lett. 2003 Apr. 25; 221(2):155-9; Plant Physiol.1994 June; 105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12):163-7(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March; 81(5):1561-5(Anabaena (various strains)); Proc Natl Acad Sci USA. 2001 Mar. 27;98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March;216(1):175-7 (various species); Mol Microbiol, 2002 June; 44(6):1517-31and Plasmid, 1993 September; 30(2):90-105 (Fremyella diplosiphon); Hallet al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al.(1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet.19: 317-322 (Chlorella); for additional promoters see also table 1 fromU.S. Pat. No. 6,027,900).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Preferred promoters include promoterssuch as RBCS2 from Chlamydomonas reinhardtii and viral promoters, suchas cauliflower mosaic virus (CMV) and chlorella virus, which have beenshown to be active in multiple species of microalgae (see for examplePlant Cell Rep. 2005 March; 23(10-11):727-35; J Microbiol. 2005 August;43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73). In otherembodiments, the Botryococcus malate dehydrogenase promoter, such anucleic acid comprising any part of SEQ ID NO:3, or the Chlamydomonasreinhardtii RBCS2 promoter (SEQ ID NO:4) can be used. Optionally, atleast 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used. Preferred promoters endogenous tospecies of the genus Chlorella are SEQ ID NO:1 and SEQ ID NO:2.

Preferred promoters useful for expression of exogenous genes inChlorella are listed in the sequence listing of this application, suchas the promoter of the Chlorella HUP1 gene (SEQ ID NO:1) and theChlorella ellipsoidea nitrate reductase promoter (SEQ ID NO:2).Chlorella virus promoters can also be used to express genes inChlorella, such as SEQ ID NOs: 1-7 of U.S. Pat. No. 6,395,965.Additional promoters active in Chlorella can be found, for example, inBiochem Biophys Res Commun. 1994 Oct. 14; 204(1):187-94; Plant Mol.Biol. 1994 October; 26(1):85-93; Virology. 2004 Aug. 15; 326(1):150-9;and Virology. 2004 Jan. 5; 318(1):214-23.

C. Selectable Markers

Any of a wide variety of selectable markers can be employed in atransgene construct useful for transforming Chlorella. Examples ofsuitable selectable markers include the nitrate reductase gene, thehygromycin phosphotransferase gene (HPT), the neomycinphosphotransferase gene, and the ble gene, which confers resistance tophleomycin. Methods of determining sensitivity of microalgae toantibiotics are well known. For example, Mol Gen Genet. 1996 Oct. 16;252(5):572-9.

More specifically, Dawson et al. (1997), Current Microbiology 35:356-362(incorporated by reference herein in its entirety), described the use ofthe nitrate reductase (NR) gene from Chlorella vulgaris as a selectablemarker for NR-deficient Chlorella sorokiniana mutants. Kim et al.(2002), Mar. Biotechnol. 4:63-73 (incorporated by reference herein inits entirety), disclosed the use of the HPT gene as a selectable markerfor transforming Chlorella ellipsoidea. Huang et al. (2007), Appl.Microbiol. Biotechnol. 72:197-205 (incorporated by reference herein inits entirety), reported on the use of Sh ble as a selectable marker forChlorella sp. DT.

D. Inducible Expression

The present invention also provides for the use of an inducible promoterto express a gene of interest. In particular, the use of an induciblepromoter to express a lipase gene permits production of the lipase aftergrowth of the microorganism when conditions have been adjusted, ifnecessary, to enhance transesterification, for example, after disruptionof the cells, reduction of the water content of the reaction mixture,and/or addition sufficient alcohol to drive conversion of TAGs to fattyacid esters.

Inducible promoters useful in the invention include those that mediatetranscription of an operably linked gene in response to a stimulus, suchas an exogenously provided small molecule (e.g., glucose, as in SEQ IDNO:1), temperature (heat or cold), light, etc. Suitable promoters canactivate transcription of an essentially silent gene or upregulate,preferably substantially, transcription of an operably linked gene thatis transcribed at a low level. In the latter case, the level oftranscription of the lipase preferably does not significantly interferewith the growth of the microorganism in which it is expressed.

Expression of transgenes in Chlorella can be performed inducibly throughpromoters such as the promoter that drives the Chlorella hexosetransporter gene (SEQ ID NO:1). This promoter is strongly activated bythe presence of glucose in the culture media.

E. Expression of Two or More Exogenous Genes

Further, a genetically engineered microorganism, such as a microalgae,may comprise and express two or more exogenous genes, such as, forexample, a lipase and a lytic gene, e.g., one encoding apolysaccharide-degrading enzyme. One or both genes can be expressedusing an inducible promoter, which allows the relative timing ofexpression of these genes to be controlled to enhance the lipid yieldand conversion to fatty acid esters. Expression of the two or moreexogenous genes may be under control of the same inducible promoter orunder control of a different inducible promoters. In the lattersituation, expression of a first exogenous gene can be induced for afirst period of time (during which expression of a second exogenous genemay or may not be induced) and expression of a second exogenous gene canbe induced for a second period of time (during which expression of afirst exogenous gene may or may not be induced). Provided herein arevectors and methods for engineering lipid-producing microbes tometabolize sucrose, which is an advantageous trait because it allows theengineered cells to convert sugar cane feedstocks into lipids.

Also provided herein are genetically engineered strains of microbes(e.g., microalgae, oleaginous yeast, bacteria, or fungi) that expresstwo or more exogenous genes, such as, for example, a fatty acyl-ACPthioesterase and a fatty acyl-CoA/aldehyde reductase, the combinedaction of which yields an alcohol product. Further provided are othercombinations of exogenous genes, including without limitation, a fattyacyl-ACP thioesterase and a fatty acyl-CoA reductase to generatealdehydes. In addition, this application provides for the combination ofa fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and a fattyaldehyde decarbonylase to generate alkanes. One or more of the exogenousgenes can be expressed using an inducible promoter.

Examples of further modifications suitable for use in the presentinvention are include genetically engineering strains of microalgae toexpress two or more exogenous genes, one encoding a transporter of afixed carbon source (such as sucrose) and a second encoding a sucroseinvertase enzyme. The resulting fermentable organisms producehydrocarbons at lower manufacturing cost than what has been obtainableby previously known methods of biological hydrocarbon production.Insertion of the two exogenous genes described above can be combinedwith the disruption of polysaccharide biosynthesis through directedand/or random mutagenesis, which steers ever greater carbon flux intohydrocarbon production. Individually and in combination, trophicconversion, engineering to alter hydrocarbon production and treatmentwith exogenous enzymes alter the hydrocarbon composition produced by amicroorganism. The alteration can be a change in the amount ofhydrocarbons produced, the amount of one or more hydrocarbon speciesproduced relative to other hydrocarbons, and/or the types of hydrocarbonspecies produced in the microorganism. For example, microalgae can beengineered to produce a higher amount and/or percentage of TAGs.

F. Compartmentalized Expression

The present invention also provides for compartmentalized expression ofa gene of interest. In particular, it can be advantageous, in particularembodiments, to target expression of the lipase to one or more cellularcompartments, where it is sequestered from the majority of cellularlipids until initiation of the transesterification reaction. Preferredorganelles for targeting are chloroplasts, mitochondria, and endoplasmicreticulum.

1. Expression in Chloroplasts

In one embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to chloroplasts. Methods fortargeting expression of a heterologous gene to the chloroplast are knownand can be employed in the present invention. Methods for targetingforeign gene products into chloroplasts are described in Shrier et al.,EMBO J. (1985) 4:25 32. See also Tomai et al. Gen. Biol. Chem. (1988)263:15104 15109 and U.S. Pat. No. 4,940,835 for the use of transitpeptides for translocating nuclear gene products into the chloroplast.Methods for directing the transport of proteins to the chloroplast arealso reviewed in Kenauf TIBTECH (1987) 5:40 47. Chloroplast targetingsequences endogenous to Chlorella are known, such as genes in theChlorella nuclear genome that encode proteins that are targeted to thechloroplast; see for example GenBank Accession numbers AY646197 andAF499684.

Wageningen UR-Plant Research International sells an IMPACTVECTOR1.4vector, which uses the secretion signal of the Chrysanthemum morifoliumsmall subunit protein to deliver a heterologous protein into thechloroplast stroma (cytoplasmic) environment, shuttling across a doublemembrane system. The protein is fused to the first 11 amino acids of themature rubisco protein in order to allow proper processing of the signalpeptide (Wong et al., Plant Molecular Biology 20: 81-93 (1992)). Thesignal peptide contains a natural intron from the RbcS gene.

In another approach, the chloroplast genome is genetically engineered toexpress the heterologous protein. Stable transformation of chloroplastsof Chlamydomonas reinhardtii (a green alga) using bombardment ofrecipient cells with high-velocity tungsten microprojectiles coated withforeign DNA has been described. See, for example, Boynton et al.,Science (1988) 240: 1534 1538; Blowers et al. Plant Cell (1989) 1:123132 and Debuchy et al., EMBO J. (1989) δ: 2803 2809. The transformationtechnique, using tungsten microprojectiles, is described by Klein etal., Nature (London) (1987) 7:70 73. Other methods of chloroplasttransformation for both plants and microalgae are known. See for exampleU.S. Pat. Nos. 5,693,507; 6,680,426; and Plant Physiol. 2002 May;129(1):7-12; and Plant Biotechnol J. 2007 May; 5(3):402-12.

As described in U.S. Pat. No. 6,320,101 (issued Nov. 20, 2001 to Kaplanet al.; which is incorporated herein by reference), cells can bechemically treated so as to reduce the number of chloroplasts per cellto about one. Then, the heterologous nucleic acid can be introduced intothe cells via particle bombardment with the aim of introducing at leastone heterologous nucleic acid molecule into the chloroplasts. Theheterologous nucleic acid is selected such that it is integratable intothe chloroplast's genome via homologous recombination which is readilyeffected by enzymes inherent to the chloroplast. To this end, theheterologous nucleic acid includes, in addition to a gene of interest,at least one nucleic acid sequence that is derived from thechloroplast's genome. In addition, the heterologous nucleic acidtypically includes a selectable marker. Further details relating to thistechnique are found in U.S. Pat. Nos. 4,945,050 and 5,693,507 which areincorporated herein by reference. A polypeptide can thus be produced bythe protein expression system of the chloroplast.

U.S. Pat. No. 7,135,620 (issued Nov. 14, 2006 to Daniell et al.;incorporated herein by reference) describes chloroplast expressionvectors and related methods. Expression cassettes are DNA constructsincluding a coding sequence and appropriate control sequences to providefor proper expression of the coding sequence in the chloroplast. Typicalexpression cassettes include the following components: the 5′untranslated region from a microorganism gene or chloroplast gene suchas psbA which will provide for transcription and translation of a DNAsequence encoding a polypeptide of interest in the chloroplast; a DNAsequence encoding a polypeptide of interest; and a translational andtranscriptional termination region, such as a 3′ inverted repeat regionof a chloroplast gene that can stabilize RNA of introduced genes,thereby enhancing foreign gene expression. The cassette can optionallyinclude an antibiotic resistance gene.

Typically, the expression cassette is flanked by convenient restrictionsites for insertion into an appropriate genome. The expression cassettecan be flanked by DNA sequences from chloroplast DNA to facilitatestable integration of the expression cassette into the chloroplastgenome, particularly by homologous recombination. Alternatively, theexpression cassette may remain unintegrated, in which case, theexpression cassette typically includes a chloroplast origin ofreplication, which is capable of providing for replication of theheterologous DNA in the chloroplast.

The expression cassette generally includes a promoter region from a genecapable of expression in the chloroplast. The promoter region mayinclude promoters obtainable from chloroplast genes, such as the psbAgene from spinach or pea, or the rbcL and atpB promoter region frommaize and rRNA promoters. Examples of promoters are described inHanley-Bowdoin and Chua, TIBS (1987) 12:67 70; Mullet et al., PlantMolec Biol. (1985) 4: 39 54; Hanley-Bowdoin (1986) PhD. Dissertation,the Rockefeller University; Krebbers et al., Nucleic Acids Res. (1982)10: 4985 5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251 3270;and Zurawski et al., Proc. Nat'l Acad. Sci. U.S.A. (1982) 79: 7699 7703.Other promoters can be identified and the relative strength of promotersso identified evaluated, by placing a promoter of interest 5′ to apromoterless marker gene and observing its effectiveness relative totranscription obtained from, for example, the promoter from the psbAgene, a relatively strong chloroplast promoter. The efficiency ofheterologous gene expression additionally can be enhanced by any of avariety of techniques. These include the use of multiple promotersinserted in tandem 5′ to the heterologous gene, for example a doublepsbA promoter, the addition of enhancer sequences and the like.

Numerous promoters active in the Chlorella chloroplast can be used forexpression of exogenous genes in the Chlorella chloroplast, such asthose found in GenBank accession number NC_(—)001865 (Chlorella vulgarischloroplast, complete genome),

Where it is desired to provide for inducible expression of theheterologous gene, an inducible promoter and/or a 5′ untranslated regioncontaining sequences which provide for regulation at the level oftranscription and/or translation (at the 3′ end) may be included in theexpression cassette. For example, the 5′ untranslated region can be froma gene wherein expression is regulatable by light. Similarly, 3′inverted repeat regions could be used to stabilize RNA of heterologousgenes. Inducible genes may be identified by enhanced expression inresponse to a particular stimulus of interest and low or absentexpression in the absence of the stimulus. For example, alight-inducible gene can be identified where enhanced expression occursduring irradiation with light, while substantially reduced expression orno expression occurs in low or no light. Light regulated promoters fromgreen microalgae are known (see for example Mol Genet Genomics. 2005December; 274(6):625-36).

The termination region which is employed will be primarily one ofconvenience, since the termination region appears to be relativelyinterchangeable among chloroplasts and bacteria. The termination regionmay be native to the transcriptional initiation region, may be native tothe DNA sequence of interest, or may be obtainable from another source.See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.

The expression cassettes may be transformed into a plant cell ofinterest by any of a number of methods. These methods include, forexample, biolistic methods (See, for example, Sanford, Trends InBiotech. (1988) 6:299 302, U.S. Pat. No. 4,945,050; electroporation(Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985) 82:5824 5828); useof a laser beam, microinjection or any other method capable ofintroducing DNA into a chloroplast.

Additional descriptions of chloroplast expression vectors suitable foruse in microorganisms such as microalgae are found in U.S. Pat. No.7,081,567 (issued Jul. 25, 2006 to Xue et al.); U.S. Pat. No. 6,680,426(issued Jan. 20, 2004 to Daniell et al.); and U.S. Pat. No. 5,693,507(issued Dec. 2, 1997 to Daniell et al.).

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe chloroplast using chloroplast targeting signals. Chloroplasttargeting sequences endogenous to Chlorella are known, such as genes inthe Chlorella nuclear genome that encode proteins that are targeted tothe chloroplast; see for example GenBank Accession numbers AY646197 andAF499684. Proteins can also be expressed in the Chlorella chloroplast byinsertion of genes directly into the chloroplast genome. Chloroplasttransformation typically occurs through homologous recombination, andcan be performed if chloroplast genome sequences are known for creationof targeting vectors (see for example the complete genome sequence of aChlorella chloroplast; Genbank accession number NC_(—)001865). Seeprevious sections herein for details of chloroplast transformation.

2. Expression in Mitochondria

In another embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to mitochondria. Methods fortargeting foreign gene products into mitochondria (Boutry et al. Nature(London) (1987) 328:340 342) have been described, including in greenmicroalgae (see for example Mol Gen Genet. 1993 January;236(2-3):235-44).

For example, an expression vector encoding a suitable secretion signalcan target a heterologous protein to the mitochondrion. TheIMPACTVECTOR1.5 vector, from Wageningen UR-Plant Research International,uses the yeast CoxIV secretion signal, which was shown to deliverproteins in the mitochondrial matrix. The protein is fused to the first4 amino acids of the yeast CoxIV protein in order to allow properprocessing of the signal peptide (Kohler et al. Plant J 11: 613-621(1997)). Other mitochondrial targeting sequences are known, includingthose functional in green microalgae. For example, see FEBS Lett. 1990Jan. 29; 260(2):165-8; and J Biol Chem. 2002 Feb. 22; 277(8):6051-8.

Proteins expressed in the nuclear genome of Chlorella can be targeted tothe mitochondria using mitochondrial targeting signals. See previoussections herein for details of mitochondrial protein targeting andtransformation.

3. Expression in Endoplasmic Reticulum

In another embodiment of the present invention, the expression of apolypeptide in a microorganism is targeted to the endoplasmic reticulum.The inclusion of an appropriate retention or sorting signal in anexpression vector ensure that proteins are retained in the endoplasmicreticulum (ER) and do not go downstream into Golgi. For example, theIMPACTVECTOR1.3 vector, from Wageningen UR-Plant Research International,includes the well known KDEL retention or sorting signal. With thisvector, ER retention has a practical advantage in that it has beenreported to improve expression levels 5-fold or more. The main reasonfor this appears to be that the ER contains lower concentrations and/ordifferent proteases responsible for post-translational degradation ofexpressed proteins than are present in the cytoplasm. ER retentionsignals functional in green microalgae are known. For example, see ProcNatl Acad Sci USA. 2005 Apr. 26; 102(17):6225-30.

G. Transformation

Cells can be transformed by any suitable technique including, e.g.,biolistics, electroporation, glass bead transformation and siliconcarbide whisker transformation. See, e.g., Examples herein.

Any convenient technique for introducing a transgene into Chlorella canbe employed in the present invention. Dawson et al. (1997) (supra)described the use of micro-projectile bombardment to introduce thenitrate reductase (NR) gene from Chlorella vulgaris into NR-deficientChlorella sorokiniana mutants, resulting in stable transformants.Briefly, 0.4 micron tungsten beads were coated with plasmid; 3×10⁷ C.sorokiniana cells were spread in the center third of a non-selectiveagar plate and bombarded with the PDS-1000/He Biolistic ParticleDelivery® system (Bio-Rad).

A preferred method for introducing a transgene into Chlorella is themethod described by Kim et al. (2002), Mar. Biotechnol. 4:63-73. Kimreports the transformation of Chlorella ellipsoidea protoplasts usingCaCl₂ and polyethylene glycol (PEG). In particular, protoplasts wereprepared by growing C. ellipsoidea cells to a density of 1-2×10⁸/Ml.Cells were recovered and washed by centrifugation for 5 minutes at 1600g and resuspended in 5 Ml of phosphate buffer (Ph 6.0) containing 0.6 Msorbitol, 0.6 M mannitol, 4% (weight/volume) cellulose (Calbiochem), 2%(weight/volume) macerase (Calbiochem), and 50 units pectinase (Sigma).The cell suspension was incubated at 25° C. for 16 hours in the darkwith gentle shaking. The resultant protoplasts were recovered bycentrifugation at 400 g for 5 minutes. The pellet was gently resuspendedin 5 Ml of f/2 medium containing 0.6 M sorbitol and 0.6 M mannitol andcentrifuged at 400 g for 5 minutes. This pellet was resuspended in 1 Mlof 0.6 M sorbitol/mannitol solution containing 50 mM CaCl₂. Then, 5 mgof transgene DNA was added, along with 25 μg calf thymus DNA (Sigma), to10⁷-10⁸ protoplasts in 0.4 Ml. After 15 minutes at room temperature, 200μL of PNC (40% polyethylene glycol 4000, 0.8 M NaCl, 50 Mm CaCl₂) wasadded and mixed gently for 30 minutes at room temperature. After this,0.6 Ml of f/2 medium supplemented with 0.6 M sorbitol/mannitol solution,1% yeast extract and 1% glucose was added, and the transformed cellswere incubated at 25° C. for 12 hours in the dark for cell wallregeneration. A similar method was used by Huang et al. (2007) (supra)to introduce a transgene encoding mercuric reductase into Chlorella sp.DT.

Electorporation has also been employed to transform Chlorella. Asreported by Maruyama et al. (2004), Biotechnology Techniques 8:821-826(incorporated by reference herein in its entirety), this technique wasused to introduce a transgene into protoplasts of Chlorellasaccharophila c-211-1a prepared from the cells in the stationary phase.Transient expression of the introduced plasmid was observed under afield strength of between 600 and 900 V/cm, and a pulse duration ofaround 400 ms, where high membrane permeability to 70-kDa FITC-dextranwas ascertained.

Examples of expression of transgenes in Chlorella can be found in theliterature (see for example Current Microbiology Vol. 35 (1997), pp.356-362; Sheng Wu Gong Cheng Xue Bao. 2000 July; 16(4):443-6; CurrentMicrobiology Vol. 38 (1999), pp. 335-341; Appl Microbiol Biotechnol(2006) 72: 197-205; Marine Biotechnology 4, 63-73, 2002; CurrentGenetics 39:5, 365-370 (2001); Plant Cell Reports 18:9, 778-780, (1999);Biologia Plantarium 42(2): 209-216, (1999); Plant Pathol. J 21(1):13-20, (2005)). Also see Examples herein.

Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowialipolytica) can be found in the literature (see, for example, Bordes etal., J Microbiol Methods, June 27 (2007)). Examples of expression oftransgenes in fungi (e.g., Mortierella alpine, Mucor circinelloides, andAspergillus ochraceus) can also be found in the literature (see, forexample, Microbiology, July; 153(Pt. 7):2013-25 (2007); Mol GenetGenomics, June; 271(5):595-602 (2004); Curr Genet, March; 21(3):215-23(1992); Current Microbiology, 30(2):83-86 (1995); Sakuradani, NISRResearch Grant, “Studies of Metabolic Engineering of UsefulLipid-producing Microorganisms” (2004); and PCT/JP2004/012021). Examplesof expression of exogenous genes in bacteria such as E. coli are wellknown; see for example Molecular Cloning: A Laboratory Manual, Sambrooket al. (3d edition, 2001, Cold Spring Harbor Press.

Vectors for transformation of microorganisms in accordance with thepresent invention can be prepared by known techniques familiar to thoseskilled in the art. The nucleotide sequence of the construct used fortransformation of multiple Chlorella species corresponds to SEQ IDNO:25. In one embodiment, an exemplary vector design for expression of alipase gene in a microorganism such as a microalgae contains a geneencoding a lipase in operable linkage with a promoter active inmicroalgae. Alternatively, if the vector does not contain a promoter inoperable linkage with the gene of interest, the gene can be transformedinto the cells such that it becomes operably linked to an endogenouspromoter at the point of vector integration. The promoterless method oftransformation has been proven to work in microalgae (see for examplePlant Journal 14:4, (1998), pp. 441-447). The vector can also contain asecond gene that encodes a protein that, e.g., imparts resistance to anantibiotic or herbicide, i.e., a selectable marker. Optionally, one orboth gene(s) is/are followed by a 3′ untranslated sequence containing apolyadenylation signal. Expression cassettes encoding the two genes canbe physically linked in the vector or on separate vectors.Co-transformation of microalgae can also be used, in which distinctvector molecules are simultaneously used to transform cells (see forexample Protist 2004 December; 155(4):381-93). The transformed cells canbe optionally selected based upon the ability to grow in the presence ofthe antibiotic or other selectable marker under conditions in whichcells lacking the resistance cassette would not grow.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

X. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1

Chlorella strains from the University of Texas culture collection weretested for growth on glycerol and glucose. The following Chlorellaspecies and strains were cultured: Chlorella kessleri (strains 263, 397,398, 2228); Chlorella sorokiniana (strains 1663, 1665, 1669, 1671,1810); Chlorella saccharophila (2911; 2469); Chlorella protothecoides(31, 249, 250, 264). Each strain was inoculated from solid media into 25ml liquid base media (2 g/L yeast extract, 2.94 mM NaNO₃, 0.17 mMCaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.4 mM K₂HPO₄, 1.28 mM KH₂PO₄, 0.43 mMNaCl) and grown shaking at 27° C. for 72 hours under a light intensityof 75 μEm⁻²s⁻¹. These cultures were used to inoculate each strain to afinal density of 1×10⁵ cells per ml into 24-well plates containing 2 mlof (a) base media only; (b) base media plus 0.1% glucose; and (c) basemedia plus 0.5% reagent grade glycerol (EM Science, catalog #GX0185-6).Plates were placed in the dark and grown for 72 hours shaking at 27° C.Samples of each strain grown in the three conditions were diluted 1.9:1in distilled H2O and absorbance was read at 600 nm in a MolecularDevices SpectraMax 340PC. All strains exhibited growth in the presenceof glucose and glycerol compared to only base media.

Example 2 Strains and Media

Chlorella protothecoides #1 (STRAIN 250), #2 (STRAIN 264) and Chlorellakessleri #1 (STRAIN 398) were obtained from the Culture Collection ofAlgae at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium. Modified Proteose mediumconsisted (g/L) of 0.25 g NaNO₃, 0.09 g K₂HPO₄, 0.175 g KH₂PO₄ 0.025 g,0.025 g CaCl₂.2H₂O, 0.075 g MgSO₄.7H₂O, and 2 g yeast extract per liter.Glycerol wastes from biodiesel production (acidulated glycerol (AG) andnon-acidulated glycerol (NAG)) were obtained from Imperial WesternProducts (Selma, Calif., USA). “Pure” or “reagent grade” glycerol wasfrom EM Science (a division of Merck KGA), catalog #GX0185-6.

Experimental Design and Growth Measurement:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol

2. Proteose+1% acidulated glycerol

3. Proteose+1% non-acidulated glycerol

4. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

5. Proteose+1% acidulated glycerol+1% glucose (added after 72 hr)

6. Proteose+1% non-acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to different media to 5×15 cells/mlconcentration. The cultures were kept in dark and were agitated byorbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr ofinitial growth, 1% (w/v) glucose was added to samples #4, 5, and 6 andcultured another 24 hr. To measure dry cell weight, 1 ml of each culturewas pelleted by centrifugation at 5,000 rpm for 5 min in an Eppendorf5415C centrifuge. After removing supernatant, cell pellets were frozenat −80° C. and lyophilized in a lab scale freeze dryer (Labconco, Mo.,USA). Results are shown in FIG. 1.

Example 3 Strains and Media

Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249) and Chlorellakessleri #2 (strain 397) were obtained from the Culture Collection ofAlgae at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Growth Measurement:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose

2. Proteose+1% acidulated glycerol+1% glucose

3. Proteose+1% non-acidulated glycerol+1% glucose

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 96 hr, cell growthwas measured for dry cell weight (see EXAMPLE 2). Results are shown inFIG. 2.

Example 4 Strains and Media

Chlorella protothecoides #3 (STRAIN 249), #4 (STRAIN 31), and Chlorellakessleri #2 (STRAIN 397) were obtained from the Culture Collection ofAlga at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium (see EXAMPLE 2)

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose

2. Proteose+1% acidulated glycerol+1% glucose

3. Proteose+1% non-acidulated glycerol+1% glucose

Each strain was inoculated to media containing different glycerols(pure, acidulated, or non-acidulated) to 5×10⁵ cells/ml concentration.The cultures were kept in dark and agitated by orbital shaker fromLabnet (Berkshire, UK) at 430 rpm. After 96 hr, lipid contents weremeasured. To measure the amount of lipid content in cells, 100 μl ofcultures were collected and washed once with same volume of media. Toeach tube, 5 μl of washed cells and 200 μl of sulfuric acid 18 M wereadded. The tubes were incubated at 90° C. in a water bath for 30 min,and 1 ml of phosphoric acid-vanillin reagent was added to the tubes andincubated at 37° C. for 15 min. To prepare the phosphoric acid-vanillinreagent, 0.12 g of vanillin was added to 20 ml of water, and the volumeadjusted to 100 ml with 85% phosphoric acid. The optical density at 530nm was read in a glass cuvette against a reference tube with 5 μl wateras sample. Results are shown in FIG. 3.

Example 5 Strains and Media

Chlorella protothecoides #2 (STRAIN 264) and Chlorella kessleri #1(STRAIN 398) were obtained from the Culture Collection of Alga at theUniversity of Texas (Austin, Tex., USA). The stock cultures weremaintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of the following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol

2. Proteose+1% non-acidulated glycerol

3. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

4. Proteose+1% non-acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to media containing different glycerols (pureor non-acidulated) to 5×10⁵ cells/ml concentration. The cultures werekept in dark and agitated by orbital shaker from Labnet (Berkshire, UK)at 430 rpm. After 72 hr of initial growth, 1% glucose was added tosample #3 and #4 and cultured another 24 hr. Lipid contents weremeasured in all samples (see EXAMPLE 4). The optical density at 600 nmwas also measured to check for non-specific absorbance and subtractedfrom O.D. 530 nm to calculate the amount of lipid. The reference curveis composed of Triolein dissolved in chloroform ranging from 1 to 10 μg.Results are shown in FIG. 4.

Example 6 Strains and Media

Chlorella protothecoides #3 (STRAIN 249) and Chlorella kessleri #2(STRAIN 397) were obtained from the Culture Collection of Alga at theUniversity of Texas (Austin, Tex., USA). The stock cultures weremaintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

2. Proteose+1% acidulated glycerol+1% glucose (added after 72 hr)

3. Proteose+1% non-acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to media containing different glycerols(pure, acidulated, or non-acidulated) to 5×10⁵ cells/ml concentration.The cultures were kept in dark and agitated by orbital shaker fromLabnet (Berkshire, UK) at 430 rpm. After 72 hr of initial growth, 1%glucose was added and cultured another 24 hr. Dried cell-weight andlipid content were measured in all samples (see EXAMPLES 2 and 5). Thelipid percentage was calculated from total lipid amount divided by driedcell weight. Results are shown in FIG. 5.

Example 7 Strains and Media

Chlorella protothecoides #2 (STRAIN 264) and Chlorella kessleri #1(STRAIN 398) were obtained from the Culture Collection of Alga at theUniversity of Texas (Austin, Tex., USA). The stock cultures weremaintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

2. Proteose+1% non-acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated media containing either 1% pure or 1%non-acidulated glycerol to 5×10⁵ cells/ml concentration. The cultureswere kept in dark and agitated by orbital shaker from Labnet (Berkshire,UK) at 430 rpm. After 72 hr of initial growth, 1% glucose was added andcultured another 24 hr. Dried cell-weight and lipid content weremeasured in all samples (see EXAMPLE 1 and 4). The lipid percentage wascalculated from total lipid amount divided by dried cell weight. Resultsare shown in FIG. 6.

Example 8 Strains and Media

Chlorella protothecoides #1 (STRAIN 250), #4 (STRAIN 31) and Chlorellakessleri #2 (STRAIN 397) were obtained from the Culture Collection ofAlga at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium (see EXAMPLE 2)

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+2% glucose

2. Proteose+1% glycerol+1% glucose

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 96 hr of initialgrowth, lipid contents were measured (see EXAMPLE 5). Results are shownin FIG. 7.

Example 9 Strains and Media

Chlorella protothecoides #3 (STRAIN 249), #4 (STRAIN 31) and Chlorellakessleri #1 (STRAIN 398) were obtained from the Culture Collection ofAlga at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+2% glucose

2. Proteose+1% glycerol+1% glucose

3. Proteose+1% glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr of initialgrowth, 1% (w/v) glucose was added to #3 media and cultured another 24hr. Dried cell-weight and lipid contents were measured in all samples(see EXAMPLES 2 and 5). The lipid percentage was calculated from totallipid amount divided by dried cell weight. Results are shown in FIG. 8.

Example 10 Strains and Media

Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249), and Chlorellakessleri #2 (STRAIN 397) were obtained from the Culture Collection ofAlga at the University of Texas (Austin, Tex., USA). The stock cultureswere maintained on modified Proteose medium (see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose

2. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

3. Proteose+1% acidulated glycerol+1% glucose

4. Proteose+1% acidulated glycerol+1% glucose (added after 72 hr)

5. Proteose+1% non-acidulated glycerol+1% glucose

6. Proteose+1% non-acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr of initialgrowth, 1% (w/v) glucose was added to #2, #4, and #6 media and culturedanother 24 hr. Lipid contents were measured in all samples (see EXAMPLE4). Results are shown in FIG. 9.

Example 11 Strains and Media

Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249), #4 (STRAIN31) and Chlorella kessleri #2 (STRAIN 397) were obtained from theCulture Collection of Alga at the University of Texas (Austin, Tex.,USA). The stock cultures were maintained on modified Proteose medium(see EXAMPLE 2).

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

1. Proteose+1% pure glycerol+1% glucose

2. Proteose+1% pure glycerol+1% glucose (added after 72 hr)

3. Proteose+1% acidulated glycerol+1% glucose

4. Proteose+1% acidulated glycerol+1% glucose (added after 72 hr)

5. Proteose+1% non acidulated glycerol+1% glucose

6. Proteose+1% non acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr of initialgrowth, 1% (w/v) glucose was added to #2, #4, and #6 media and culturedanother 24 hr. Dried cell-weight was measured in all samples (seeEXAMPLE 2). Results are shown in FIG. 10.

Example 12 Vector Construction

A BamHI-SacII fragment containing the CMV promoter, a hygromycinresistance cDNA, and a CMV 3′ UTR (SEQ ID NO:5, a subsequence of thepCAMBIA1380 vector, Cambia, Canberra, Australia) was cloned into theBamHI and SacII sites of pBluescript and is referred to herein as pHyg.

Biolistic Transformation of Chlorella

S550d gold carriers from Seashell Technology were prepared according tothe protocol from manufacturer. Linearized pHyg plasmid (20 μg) wasmixed with 50 μl of binding buffer and 60 μl (30 mg) of S550d goldcarriers and incubated in ice for 1 min. Precipitation buffer (100 μl)was added, and the mixture was incubated in ice for another 1 min. Aftervortexing, DNA-coated particles were pelleted by spinning at 10,000 rpmin an Eppendorf 5415C microfuge for 10 seconds. The gold pellet waswashed once with 500 μl of cold 100% ethanol, pelleted by brief spinningin the microfuge, and resuspended with 50 μl of ice-cold ethanol. Aftera brief (1-2 sec) sonication, 10 μl of DNA-coated particles wereimmediately transferred to the carrier membrane.

Chlorella protothecoides culture (University of Texas Culture Collection250) was grown in proteose medium (2 g/L yeast extract, 2.94 mM NaNO₃,0.17 mM CaCl2.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.4 mM K2HPO4, 1.28 mM KH2PO4,0.43 mM NaCl) on a gyratory shaker under continuous light at 75 μmolphotons m⁻² sec⁻¹ until it reached a cell density of 2×10⁶ cells/ml. Thecells were harvested, washed once with sterile distilled water, andresuspended in 50 μl of medium. 1×10⁷ cells were spread in the centerthird of a non-selective proteose media plate. The cells were bombardedwith the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad).Rupture disks (1100 and 1350 psi) were used, and the plates were placed9 and 12 cm below the screen/macrocarrier assembly. The cells wereallowed to recover at 25° C. for 12-24 h. Upon recovery, the cells werescraped from the plates with a rubber spatula, mixed with 100 μl ofmedium and spread on hygromycin contained plates (200 μg/ml). After 7-10days of incubation at 25° C., colonies representing transformed cellswere visible on the plates from 1100 and 1350 psi rupture discs and from9 and 12 cm distances. Colonies were picked and spotted on selectiveagar plates for a second round of selection.

Transformation of Chlorella by Electroporation

Chlorella protothecoides culture was grown in proteose medium on agyratory shaker under continuous light at 75 μmol photons m⁻² sec⁻¹until it reached a cell density of 2×10⁶ cells/ml. The cells wereharvested, washed once with sterile distilled water, and resuspended ina tris-phosphate buffer (20 m M Tris-HCl, pH 7.0; 1 mM potassiumphosphate) containing 50 mM sucrose to a density of 4×10⁸ cells/ml.About 250 μl cell suspension (1×10⁸ cells) was placed in a disposableelectroporation cuvette of 4 mm gap. To the cell suspension, 5 μg oflinearized pHyg plasmid DNA and 200 μg of carrier DNA (sheared salmonsperm DNA) was added. The electroporation cuvette was then incubated ina water bath at 16° C. for 10 minutes. An electrical pulse (1100 V/cm)was then applied to the cuvette at a capacitance of 25 μF (no shuntresistor was used for the electroporation) using a Gene Pulser II(Bio-Rad Labs, Hercules, Calif.) electroporation apparatus. The cuvettewas then incubated at room temperature for 5 minutes, following whichthe cell suspension was transferred to 50 ml of proteose media, andshaken on a gyratory shaker for 2 days. Following recovery, the cellswere harvested by centrifugation at low speed, resuspended in proteosemedia, and plated at low density on plates supplemented with 200 μg/mlhygromycin. The plates were incubated under continuous light at 75 μmolphotons m⁻² sec⁻¹. Transformants appeared as colonies in 1-2 weeks.Colonies were picked and spotted on selective agar plates for a secondround of selection.

Genotyping

A subset of colonies that survived a second round of selection werecultured in small volume and harvested. Pellets of approximately 5-10 uLvolume were resuspended in 50 uL of 10 mM NaEDTA by vortexing and thenincubated at 100° C. for 10. The tubes were then vortexed briefly andsonicated for 10 seconds, then centrifuged at 12,000×g for 1 minute. 2uL of supernatant as template was used in a 50 uL PCR reaction. Primersused for genotyping were SEQ ID NO:6 and SEQ ID NO:7. PCR conditionswere as follows: 95° C. 5 min×1 cycle; 95° C. 30 sec-58° C. 30 sec-72°C. 1 min 30 sec×35 cycles; 72° C. 10 min×1 cycle. The expected 992 bpfragment was found in 6 of 10 colonies from the biolistic method andfrom a single electroporation colony. A lower sized, nonspecific bandwas present in all lanes. Results are shown in FIG. 16. To confirm theidentity of the amplified 992 bp fragment, two biolistic bands and theelectroporation band were excised from the gel and individuallysequenced. The sequence of all three bands corresponded to the expected992 bp fragment. (DNA ladder: Bionexus® All Purpose Hi-Lo® DNA laddercatalog #BN2050).

Example 13 Strains and Media

(a) Spirulina platensis (UTEX 2340) and (b) Navicula pelliculosa (UTEX667) were obtained from the Culture Collection of Algae at theUniversity of Texas (Austin, Tex., USA). The stock culture of Spirulinawas maintained in Spirulina medium and Navicula was maintained in soilextract medium (SEM). Spirulina medium consisted of 162 mM NaHCO₃, 38 mMNa₂CO₃, 1.9 mM K₂HPO₄, 29 mM NaNO₃, 5.75 mM K₂SO₄, 17.1 mM NaCl, 0.8 mMMgSO₄.7H₂O, 0.25 mM CaCl₂.2H₂O, 2 mM Na₂EDTA, 0.36 mM FeCl₃.6H₂O, 0.21mM MnCl₂-4H₂O, 0.037 mM ZnCl₂, 0.0085 mM CoCl₂-6H₂O, 0.017 mMNaMoO₄.2H₂O, 0.78 μM CuSO₄.5H₂O, 0.15 μM ZnSO₄.7H₂O, 10 μM H₃BO₃, and0.001 mM Vitamin B₁₂. Soil extract medium consisted of 2.94 mM NaNO₃,0.17 mM CaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.43 mM K₂HPO₄, 1.29 mM KH₂PO₄,0.43 mM NaCl, and soil extract. Glycerol wastes from biodieselproduction (acidulated glycerol (AG) and non-acidulated glycerol (NAG))were obtained from Imperial Western Products (Selma, Calif., USA).

Experimental Design and Growth Measurement:

For each strain, 1 ml of following different media was prepared in24-well plates.

(a)

7. Spirulina medium+2% glucose

8. Spirulina medium+2% reagent grade glycerol

9. Spirulina medium+2% non-acidulated glycerol

10. Spirulina medium+1% non-acidulated glycerol+1% glucose

(b)

1. SEM+2% glucose

2. SEM+2% reagent grade glycerol

3. SEM+1% reagent grade glycerol+1% glucose

4. SEM+2% acidulated glycerol

5. SEM+1% acidulated glycerol+1% glucose

6. SEM+2% non-acidulated glycerol

7. SEM+1% non-acidulated glycerol+1% glucose

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 96 hr, lipidcontents were measured. To measure the amount of lipid content in cells,100 μl of cultures were collected and washed once with same volume ofmedia. To each tube, 5 μl of washed cells and 200 μl of sulfuric acid 18M were added. The tubes were incubated at 90° C. water bath for 30 min,and 1 ml of phosphoric acid-vanillin reagent were added to the tubes andincubated at 37° C. for 15 min. To prepare the phosphoric acid-vanillinreagent, 0.12 g of vanillin was added to 20 ml of water, and the volumeadjusted to 100 ml with 85% phosphoric acid. The optical density at 530nm was read in a glass cuvette against a reference tube with 5 μl wateras sample. The reference curve is composed of Triolein dissolved inchloroform ranging from 1 to 10 μg.

To measure dried cell-weight, 0.5 ml of each culture was pelleted bycentrifugation at 5000 rpm for 5 min. After removing supernatant, cellpellets were frozen at −80° C. and dried overnight in a Freeze Drysystem (Labconco, Mo., USA). The lipid percentage was calculated fromtotal lipid amount divided by dried cell weight. Results are shown inFIG. 11.

Example 14 Strains and Media

Scenedesmus armatus (UTEX 2552) was obtained from the Culture Collectionof Algae at the University of Texas (Austin, Tex., USA). The stockcultures were maintained on modified Proteose medium. Modified Proteosemedium consisted (g/L) of 0.25 g NaNO₃, 0.09 g K₂HPO₄, 0.175 g KH₂PO₄0.025 g, 0.025 g CaCl₂.2H₂O, 0.075 g MgSO₄.7H₂O, and 2 g yeast extractper liter.

Experimental Design and Growth and Lipid Measurement:

For each growth condition, 1 ml of following different media wasprepared in 24-well plates.

(a), (b)

1. Proteose+2% glucose

2. Proteose+2% glycerol

3. Proteose+2% acidulated glycerol

4. Proteose+2% non-acidulated glycerol

5. Proteose+1% non-acidulated glycerol+1% glucose

Scenedesmus armatus (UTEX 2552) was inoculated to different media to5×10⁵ cells/ml concentration. The cultures were kept in dark andagitated by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After96 hr, cell growth was measured by dried cell-weight, and lipid contentwas measured by phosphor-vanillin assay. (see EXAMPLE 13). The lipidpercentage was calculated from total lipid amount divided by dried cellweight. Results are shown in FIG. 12.

Example 15 Strains and Media

Navicula pelliculosa (UTEX 667) was obtained from the Culture Collectionof Algae at the University of Texas (Austin, Tex., USA). The stockcultures were maintained on soil extract medium (see EXAMPLE 13)

Experimental Design and Growth Measurement:

For each growth condition, 1 ml of following different media wasprepared in 24-well plates.

1. SEM+2% glucose

2. SEM+2% glycerol

3. SEM+2% acidulated glycerol

4. SEM+1% acidulated glycerol+1% glucose

5. SEM+2% non-acidulated glycerol

6. SEM+1% non-acidulated glycerol+1% glucose

Navicula pelliculosa (UTEX 667) was inoculated to media containingglucose or different glycerols (pure, acidulated, or non-acidulated) to5×10⁵ cells/ml concentration. The cultures were kept in dark andagitated by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After96 hr, cell growth was measured by dried cell-weight (see EXAMPLE 13).Results are shown in FIG. 13.

Example 16 Strains and Media

Scenedesmus armatus (UTEX 2552) and Navicula pelliculosa (UTEX 667) wereobtained from the Culture Collection of Algae at the University of Texas(Austin, Tex., USA). The stock cultures were maintained on modifiedProteose medium for Scenedesmus armatus and soil extract medium forNavicula pelliculosa (see EXAMPLE 1).

Experimental Design and Growth Measurement:

For each strain, 1 ml of following different media was prepared in24-well plates.

Scenedesmus armatus

5. Proteose+1% acidulated glycerol+1% glucose

6. Proteose+1% acidulated glycerol+1% glucose (added after 72 hr)

Navicula pelliculosa

1. SEM+1% acidulated glycerol+1% glucose

2. SEM+1% acidulated glycerol+1% glucose (added after 72 hr)

Each strain was inoculated to media to 5×10⁵ cells/ml concentration. Thecultures were kept in dark and agitated by orbital shaker from Labnet(Berkshire, UK) at 430 rpm. After 72 hr of initial growth, 1% glucosewas added to sample #2 and cultured another 24 hr. Cell growth wasmeasured by dried cell-weight (see EXAMPLE 13). Results are shown inFIG. 14.

Example 17 Strains and Media

Chlorella protothecoides (UTEX 31) was obtained from the CultureCollection of Algae at the University of Texas (Austin, Tex., USA). Thestock cultures were maintained on modified Proteose medium (see EXAMPLE1)

Experimental Design:

For each condition, 1 ml of following different media was prepared in24-well plates.

4. Proteose

5. Proteose+0.5% glucose

6. Proteose+0.5% xylose

7. Proteose+0.25% glucose+0.25% xylose

Chlorella protothecoides #4 (UTEX 31) was inoculated to media containingdifferent sugars (glucose, or xylose) to 3×10⁵ cells/ml concentration.The cultures were kept in dark and agitated by orbital shaker fromLabnet (Berkshire, UK) at 430 rpm. After 72 hr of growth, cell growthwas measured by counting cell numbers of each culture. Results are shownin FIG. 15.

Example 18

Chlorella protothecoides strains #1, #3, and #4 were obtained from theCulture Collection of Algae at the University of Texas (Austin, Tex.,USA). The stock cultures were maintained on modified Proteose medium(see EXAMPLE 1). For each condition, 1 ml of following different mediawas prepared in 24-well plates.

1. Proteose

2. Proteose+1% glucose

3. Proteose+1% fructose

Each strain was inoculated to media containing different sugars(glucose, or fructose) to 1×10⁶ cells/ml concentration. The cultureswere kept in dark and agitated by orbital shaker from Labnet (Berkshire,UK) at 430 rpm. After 96 hr of growth, cell density was measured bycounting cell numbers of each culture. Results are shown in FIG. 20.

Example 19 Chlorella on Sucrose

Materials and Methods:

Chlorella protothecoides (UTEX 249) was inoculated into three 50 mlflasks of Proteose media with 1% sucrose (2.94 mM NaNO₃, 0.428 mMK₂HPO₄, 1.28 mM KH₂PO₄, 0.427 mM NaCl, 0.17 mM CaCl₂-2H₂O, 0.3 mMMgSO₄-7H₂O, proteose peptone 1 g/L) to a final cell density of 4×10⁵cells per ml. Invertase (Sigma #I4504) was added to two of the culturesat 0.01 U/ml and 0.05 U/ml. All three cultures were grown in the darkfor ˜60 hrs shaking at 150 rpm.

Results:

Final cell counts were performed on all three cultures after ˜60 hrs ofshaking in the dark. The control flask reached 4.4×10⁵ cells per mlwhile the 0.01 U/ml and 0.05 U/ml flasks reached cell densities of 1×10⁸and 3×10⁸ respectively. Each flask was checked for contamination at theend of the experiment by microscopic analysis and all were clean.

Example 20 Chlorella Strains Growing on Sucrose

Cultures of Chlorella kessleri ((a) UTEX 397 and (b) UTEX 398) andChlorella fusca ((a) UTEX 251 and (b) UTEX 1801) were inoculated fromautotrophic liquid cultures into 10 ml of Proteose+1% sucrose media in50 ml flasks at 1×10⁶ cells/ml. Control cultures were also inoculated atthe same density with only Proteose media. Cultures were grown at 28° C.in the dark shaking at 250 rpm for 7 days, at which point cell densitywas measured by hemocytometer. As shown in FIGS. 21-22, all four strainsgrew on sucrose compared to the initial cell density and theproteose-only control.

Example 21 Chlorella protothecoides Growth on Molasses with a SucroseInvertase

Preparation of Chlorella Cells for Inoculation:

A 10 ml liquid culture of Chlorella was started taking the inoculum froma solid Proteose plate. The cultures were grown in light forapproximately 2 days at 26° C. Growth was measured using an opticaldensitometer (OD) at 750 nm and by determining dry cell weights.

Preparation of Molasses and Sugar Stock Solutions:

A 5% stock solution was prepared with glucose, sucrose and threedifferent molasses samples (labeled BS1, BS2 and HTM) obtained from thecommercial processing of sugarcane into sugar, as shown in the followingTable 13. The pH of all stocks was verified to be in the range of 6-6.6,and the stocks were then autoclaved.

TABLE 13 Molasses and sugar solutions. 5% sugar dil. in 100 mls Molasses% Sugar grams or mls HTM 78.72 6.4 BS1 (FL) 44.25 11.3 BS2 (AU) 51.559.7 Sucrose 100 5 Glucose 100 5

Preparation of Invertase Solution:

A 40 units/ml stock solution of invertase was prepared by reconstituting1 mg of a 400 unit/mg Invertase (Sigma) in 10 milliliters of distilledwater.

Experimental Conditions and Setup:

10 ml cultures were prepared, each consisting of 1% final molasses/sugarconcentration, 0.05 units/ml Invertase, and 1.0×10⁶ cells per ml ofChlorella protothecoides in a base Protease media. The cultures werenumbered as follows: (1) media only control; (2) 1% HTM; (3) 1% BS1; (4)1% BS2; (5) 1% glucose; and (6) 1% sucrose. A similar control set wasalso prepared without the addition of Invertase. The cultures were grownin darkness for five days shaking at 250 rpm at 28° C.

Results:

Growth of the Chlorella protothecoides cells was evaluated following thefive days of incubation on the respective feedstock in darkness. Asshown in FIGS. 23-24, the cells can be grown on molasses in the presenceof a sucrose invertase with yields comparable to that of growth on purereagent-grade glucose.

Example 22 Genetic Engineering of Chlorella protothecoides to Express anExogenous Sucrose Invertase

Strains and Media:

Chlorella protothecoides (UTEX 250) was obtained from the CultureCollection of Alga at the University of Texas (Austin, Tex., USA). Thestock cultures were maintained on modified Proteose medium. ModifiedProteose medium consists of 0.25 g NaNO₃, 0.09 g K₂HPO₄, 0.175 g KH₂PO₄0.025 g, 0.025 g CaCl₂.2H₂O, 0.075 g MgSO₄.7H₂O, and 2 g yeast extractper liter (g/L).

Plasmid Construction:

To express the secreted form of invertase in Chlorella protothecoides, aSaccharomyces cerevisiae SUC2 gene was placed under the control of threedifferent promoters: Cauliflower mosaic virus 35S promoter (CMV),Chlorella virus promoter (NC-1A), and Chlorella HUP1 promoter. A yeastSUC2 gene was synthesized to accommodate codon usage optimized for C.protothecoides and includes a signal sequence required for directingextracellular secretion of invertase. Each construct was built inpBluescript KS+, and EcoRI/AscI, AscI/XhoI, and XhoI/BamHI sites wereintroduced to each promoter, invertase gene, and CMV 3′UTR,respectively, by PCR amplification using specific primers. Purified PCRproducts were cloned sequentially. An illustration of the finalconstructs is shown in FIG. 25.

Transformation of Chlorella protothecoides:

A Chlorella protothecoides culture was grown in modified Proteose mediumon a gyratory shaker under continuous light at 75 μmol photons m⁻² sec⁻¹till it reached a cell density of 6×10⁶ cells/ml.

For biolistic transformation, S550d gold carriers from SeashellTechnology were prepared according to the protocol from themanufacturer. Briefly, a linearized construct (20 μg) by BsaI was mixedwith 50 μl of binding buffer and 60 μl (3 mg) of S550d gold carriers andincubated in ice for 1 min. Precipitation buffer (100 μl) was added, andthe mixture was incubated in ice for another 1 min. After mildvortexing, DNA-coated particles were pelleted by spinning at 10,000 rpmin an Eppendorf microfuge for 10 seconds. The gold pellet was washedonce with 500 μl of cold 100% ethanol, pelleted by brief spinning in themicrofuge, and resuspended with 50 μl of ice-cold ethanol. After a brief(1-2 sec) sonication, 10 μl of DNA-coated particles were immediatelytransferred to the carrier membrane. The cells were harvested, washedonce with sterile distilled water, resuspended in 50 μl of medium (1×10⁷cells), and were spread in the center third of a non-selective Proteousplate. The cells were bombarded with the PDS-1000/He Biolistic ParticleDelivery system (Bio-Rad). Rupture disks (1100 and 1350 psi) were used,and the plates were placed 9-12 cm below the screen/macrocarrierassembly. The cells were allowed to recover at 25° C. for 12-24 hours.Upon recovery, the cells were scraped from the plates with a rubberspatula, mixed with 100 μl of medium and spread on modified Proteoseplates with 1% sucrose. After 7-10 days of incubation at 25° C. in thedark, colonies representing transformed cells were visible on theplates.

For transformation with electroporation, cells were harvested, washedonce with sterile distilled water, and resuspended in a Tris-phosphatebuffer (20 m M Tris-HCl, pH 7.0; 1 mM potassium phosphate) containing 50mM sucrose to a density of 4×10⁸ cells/ml. About 250 μl cell suspension(1×10⁸ cells) was placed in a disposable electroporation cuvette of 4 mmgap. To the cell suspension, 5 μg of linearized plasmid DNA and 200 μgof carrier DNA (sheared salmon sperm DNA) were added. Theelectroporation cuvette was then incubated in an ice water bath at 16°C. for 10 min. An electrical pulse (1100 V/cm) was then applied to thecuvette at a capacitance of 25 μF (no shunt resistor was used for theelectroporation) using a Gene Pulser II (Bio-Rad Labs, Hercules, Calif.)electroporation apparatus. The cuvette was then incubated at roomtemperature for 5 minutes, following which the cell suspension wastransferred to 50 ml of modified Proteose media, and shaken on agyratory shaker for 2 days. Following recovery, the cells were harvestedat low speed (4000 rpm), resuspended in modified Proteose media, andplated out at low density on modified Proteose plates with 1% sucrose.After 7-10 days of incubation at 25° C. in the dark, coloniesrepresenting transformed cells were visible on the plates.

Screening Transformants and Genotyping:

The colonies were picked from dark grown-modified Proteose plates with1% sucrose, and approximately the same amount of cells were transferredto 24 well-plates containing 1 ml of modified Proteose liquid media with1% sucrose. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm for 5 days.

To verify the presence of the invertase gene introduced in Chlorellatransformants, DNA of each transformant was isolated and amplified witha set of gene-specific primers (CMV construct: forward primer(CAACCACGTCTTCAAAGCAA) (SEQ ID NO:6)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:9), CV constructs: forward primer(TTGTCGGAATGTCATATCAA) (SEQ ID NO:10)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:11), and HUP1 construct: forwardprimer (AACGCCTTTGTACAACTGCA) (SEQ ID NO:12)/reverse primer(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:13)). For quick DNA isolation, avolume of cells (approximately 5-10 uL in size) were resuspended in 50uL of 10 mM Na-EDTA. The cell suspension was incubated at 100° C. for 10min and sonicated for 10 sec. After centrifugation at 12000 g for 1 min,3 uL of supernatant was used for the PCR reaction. PCR amplification wasperformed in the DNA thermal cycler (Perkin-Elmer GeneAmp 9600). Thereaction mixture (50 uL) contained 3 uL extracted DNA, 100 pmol each ofthe respective primers described above, 200 uM dNTP, 0.5 units of TaqDNA polymerase (NEB), and Taq DNA polymerase buffer according to themanufacturer's instructions. Denaturation of DNA was carried out at 95°C. for 5 min for the first cycle, and then for 30 sec. Primer annealingand extension reactions were carried out at 58° C. for 30 sec and 72° C.for 1 min respectively. The PCR products were then visualized on 1%agarose gels stained with ethidium bromide. FIG. 26 shows the PCRgenotype results of C. protothecoides transformants using thegene-specific primers identified above. Arrows show the expected size ofthe PCR product, and stars represent DNA samples from each transformantshowing the PCR product matched to the expected size (V: Vector only,WT: wild-type).

Growth in Liquid Culture:

After five days growth in darkness, the genotype-positive transformantsshowed growth on minimal liquid Proteose media+1% sucrose in darkness,while wild-type cells showed no growth in the same media in darkness.

Example 23 Transformation of Algal Strains with a Secreted InvertaseDerived from S. cerevisiae

Secreted Invertase:

A gene encoding a secreted sucrose invertase (Gen Bank Accession no.NP_(—)012104 from Saccharomyces cerevisiae) was synthesized de-novo as a1599 bp Asc I-Xho fragment that was subsequently sub-cloned into a pUC19derivative possessing the Cauliflower Mosaic Virus 35s promoter and 3′UTR as EcoR I/Asc I and Xho/Sac I cassettes, respectively.

Growth of Algal Cells:

Media used in these experiments was liquid base media (see Example 1)and solid base media (+1.5% agarose) containing fixed carbon in the formof sucrose or glucose (as designated) at 1% final concentration. Thestrains used in this experiment did not grow in the dark on base mediain the absence of an additional fixed carbon source. Species were struckout on plates, and grown in the dark at 28° C. Single colonies werepicked and used to inoculate 500 mL of liquid base media containing 1%glucose and allowed to grow in the dark until mid-log phase, measuringcell counts each day. Each of the following strains had been previouslytested for growth on sucrose in the dark as a sole carbon source andexhibited no growth, and were thus chosen for transformation with asecreted invertase: (1) Chlorella protothecoides (UTEX 31); (2)Chlorella minutissima (UTEX 2341); and (3) Chlorella emersonii (CCAP211/15).

Transformation of Algal Cells via Particle Bombardment:

Sufficient culture was centrifuged to give approximately 1-5×10⁸ totalcells. The resulting pellet was washed with base media with no addedfixed carbon source. Cells were centrifuged again and the pellet wasresuspended in a volume of base media sufficient to give 5×10⁷ to 2×10⁸cells/ml. 250-1000 μl of cells were then plated on solid base mediasupplemented with 1% sucrose and allowed to dry onto the plate in asterile hood. Plasmid DNA was precipitated onto gold particles accordingto the manufacturer's recommendations (Seashell Technology, La Jolla,Calif.). Transformations were carried out using a BioRad PDS He-1000particle delivery system using 1350 psi rupture disks with themacrocarrier assembly set at 9 cm from the rupture disk holder.Following transformations, plates were incubated in the dark at 28° C.All strains generated multiple transformant colonies. Control platestransformed with no invertase insert, but otherwise prepared in anidentical fashion, contained no colonies.

Analysis of Chlorella protothecoides Transformants:

Genomic DNA was extracted from Chlorella protothecoides wild type cellsand transformant colonies as follows: Cells were resuspended in 100 ulextraction buffer (87.5 mM Tris Cl, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH8.0, 0.25% SDS) and incubated at 60° C., with occasional mixing viainversion, for 30 minutes. For PCR, samples were diluted 1:100 in 20 mMTris Cl, pH 8.0.

Genotyping was done on genomic DNA extracted from WT, the transformantsand plasmid DNA. The samples were genotyped for the marker gene. Primers2383 (5′ CTGACCCGACCTATGGGAGCGCTCTTGGC 3′) (SEQ ID NO:20) and 2279 (5′CTTGACTTCCCTCACCTGGAATTTGTCG 3′) (SEQ ID NO:21) were used in thisgenotyping PCR. The PCR profile used was as follows: 94° C. denaturationfor 5 min; 35 cycles of 94° C.-30 sec, 60° C.-30 sec, 72° C.-3 min; 72°C.-5 min. A band of identical size was amplified from the positivecontrols (plasmid) and two transformants of Chlorella protothecoides(UTEX 31), as shown in FIG. 27.

Analysis of Chlorella minutissima and Chlorella emersonii transformants:Genomic DNA was extracted from Chlorella WT and the transformants asfollows: Cells were resuspended in 100 ul extraction buffer (87.5 mMTris Cl, pH 8.0, 50 mM NaCl, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubatedat 60° C., with occasional mixing via inversion, for 30 minutes. ForPCR, samples were diluted 1:100 in 20 mM Tris Cl, pH 8.0. Genotyping wasdone on genomic DNA extracted from WT, the transformants and plasmidDNA. The samples were genotyped for the marker gene. Primers 2336 (5′GTGGCCATATGGACTTACAA 3′) (SEQ ID NO:22) and 2279(5′CTTGACTTCCCTCACCTGGAATTTGTCG 3′) (SEQ ID NO:21) were designatedprimer set 2 (1215 bp expected product), while primers 2465(5′CAAGGGCTGGATGAATGACCCCAATGGACTGTGGTACGACG 3′) (SEQ ID NO:23) and 2470(5′CACCCGTCGTCATGTTCACGGAGCCCAGTGCG 3′) (SEQ ID NO:24) were designatedprimer set 4 (1442 bp expected product). The PCR profile used was asfollows: 94° C. denaturation for 2 min; 29 cycles of 94° C.-30 sec, 60°C.-30 sec, 72° C.-1 min, 30 sec; 72° C.-5 min. A plasmid controlcontaining the secreted invertase was used as a PCR control. FIG. 28shows the transformation of the Chlorella minutissima (UTEX 2341) andChlorella emersonii (CCAP 211/15) species of microalgae with the geneencoding a secreted invertase.

The sequence of the invertase construct corresponds to SEQ ID NO:25.

Example 24 Growth of Algal Strains Compared to S. cerevisiae on aCellulosic Feedstock Prepared with Celluclast

Strains and Culture Conditions:

Algal strains used in this study are listed in Table 14 below, and weregrown in Proteose media with exogenously provided cellulosic materialand in some cases additional fixed carbon in the form of glucose. Twentyfour algal strains were used in this study, including five differentgenera encompassing eleven different species of Chlorella, two ofParachlorella and Prototheca, and one each of Bracteococcus andPseudochlorella. Saccharomyces cerevisiae (strain PJ-69-4A) was grown inYPD media (per liter, 10 g Bacto-yeast extract, 20 g Bacto peptone and20 g glucose). Both algae and yeast were grown at 28° C. in the dark.Growth of these strains on Proteose media in the dark in the absence ofcellulosic material or other additional fixed carbon either did notoccur or was extremely minimal.

Liberation of Glucose from Cellulosic Material Via EnzymaticDepolymerization Treatment:

Wet, exploded corn stover material was prepared by the NationalRenewable Energy Laboratory (Golden, Colo.) by cooking corn stover in a1.4% sulfuric acid solution and dewatering the resultant slurry. Using aMettler Toledo Moisture analyzer, the dry solids in the wet corn stoverwere determined to be 24%. A 100 g wet sample was resuspended indeionized water to a final volume of 420 ml and the pH was adjusted to4.8 using 10 N NaOH. Celluclast™ (Novozymes) (a cellulase) was added toa final concentration of 4% and the resultant slurry incubated withshaking at 50° C. for 72 hours. The pH of this material was thenadjusted to 7.5 with NaOH (negligible volume change), filter sterilizedthrough a 0.22 um filter and stored at −20° C. A sample was reserved fordetermination of glucose concentration using a hexokinase based kit fromSigma, as described below.

Determination of Glucose Concentration Liberated by Celluclast Treatmentof Wet Corn Stover:

Glucose concentrations were determined using Sigma Glucose Assay Reagent#G3293. Samples, treated as outlined above, were diluted 400 fold and 40μl was added to the reaction. The corn stover cellulosic preparation wasdetermined to contain approximately 23 g/L glucose.

TABLE 14 Algal strains grown on cellulosic feedstock. Genus/SpeciesSource/Designation Bracteococcus minor UTEX 66 Chlorella ellipsoidea SAG2141 Chlorella kessleri UTEX 1808 Chlorella kessleri UTEX 397 Chlorellaemersonii CCAP 211/15 Chlorella luteoviridis SAG 2133 Chlorellaluteoviridis SAG 2198 Chlorella luteoviridis SAG 2214 Chlorellaluteoviridis UTEX 22 Bracteococcus medionucleatus UTEX 1244 Chlorellaminutissima CCALA 20024 Chlorella minutissima UTEX 2341 Chlorella ovalisCCAP 211/21A Chlorella protothecoides CCAP 211/8d Chlorellaprotothecoides UTEX 250 Chlorella saccharophila UTEX 2911 Chlorellasorokiniana UTEX 1230 Chlorella sp. SAG 241.80 Chlorella vulgaris CCAP211/11C Parachlorella kessleri SAG 12.80 Parachlorella kessleri SAG27.87 Prototheca moriformis UTEX 1441 Prototheca moriformis UTEX 1434Pseudochlorella aquatica SAG 2149

In Table 14, and as used herein, UTEX refers to the culture collectionof algae at the University of Texas (Austin, Tex., USA), SAG refers tothe Culture Collection of Algae at the University of Göttingen(Göttingen, Germany), CCAP refers to the culture collection of algae andprotozoa managed by the Scottish Association for Marine Science(Scotland, United Kingdom) and CCALA refers to the culture collection ofalgal laboratory at the Institute of Botany (T{hacek over (r)}ebo{hacekover (n)}, Czech Republic).

Determination of Growth on Cellulosic Material: After enzymatictreatment and saccharification of cellulose to glucose, xylose, andother monosaccharide sugars, the material prepared above was evaluatedas a feedstock for the growth of 24 algal strains or S. cerevisiae inProteose media or YPD media respectively. Proteose media was made up toa final glucose concentration of 23 g/L (the final concentration ofglucose generated via cellulolytic treatment of corn stover), as was YPDfor growth of S. cerevisiae, by adding varying amounts of pure glucoseand/or depolymerized cellulosic material. Varying concentrations ofcellulosic material were included, providing 0, 12.5, 25, 50 and 100% ofthe 23 g/L glucose in each media, the components of which are shown inTable 15 below. One ml of the appropriate media was added to wells of a24 well plate. S. cerevisiae grown heterotrophically at 28° C. in YPDserved as inoculum (20 ul) for the yeast wells. Twenty microliters ofinoculum for the 24 algal strains was furnished by alga cells grownmixotrophically in Proteose media containing 20 g/L glucose.

TABLE 15 Cellulosic feedstock preparations. Vol. of 100% Vol. of YPDVol. of Proteose Cellulosics Made Vol. of 100% Final 23.1 g/L Media 23.1g/L up to Proteose Cellulosics Made Volume Percent Glucose (ml) Glucose(ml) Media (ml) up to YPD (ml) (ml) Cellulosics 0 1 0 0 1 0 0 0.8750.125 0 1 12.5 0 0.75 0.25 0 1 25 0 0.5 0.5 0 1 50 0 0 1 0 1 100 1 0 0 01 0 0.875 0 0 0.125 1 12.5 0.75 0 0 0.25 1 25 0.5 0 0 0.5 1 50 0 0 0 1 1100

Table 15 shows the volumes of depolymerized corn stover modified tocontain Proteose or YPD media components that were added to Proteose orYPD media respectively to yield medias containing the indicatedpercentage of cellulosics. Medias were prepared in order to obtain afinal glucose concentration of 23 g/L in all cases. Volume of mediaprior to the addition of either 20 μl of mid-log phase grown yeast oralga cells was 1 ml. Cells were incubated two days in the dark on thevarying concentrations of cellulosic feedstocks at 28° C. with shaking(300 rpm). Growth was assessed by measurement of absorbance at 750 nm ina UV spectrophotometer. Surprisingly, all strains grew on the cellulosicmaterial prepared with Celluclast, including media conditions in which100% of fermentable sugar was cellulosic-derived.

Example 25 Growth of 24 Algal Strains and S. cerevisiae on VariousCellulosic Feedstocks Prepared with Accellerase 1000™ and Celluclast™

Strains and Culture Conditions:

Algal strains used in this Example are listed in Table 14 (above) andwere grown in Proteose media plus additional fixed carbon in the form ofdepolymerized cellulosic material and/or pure glucose. Saccharomycescerevisiae (strain pJ69-4-a) was grown in YPD media plus additionalfixed carbon in the form of depolymerized cellulosic material and/orpure glucose. Both algae and yeast were grown at 28° C. in the dark.

Liberation of Glucose from Cellulosic Material via EnzymaticDepolymerization Treatment:

Wet, exploded corn stover material was prepared by the NationalRenewable Energy Laboratory (Golden, Colo.) by cooking corn stover in a1.4% sulfuric acid solution and dewatering the resultant slurry.Switchgrass was also prepared by The National Renewable EnergyLaboratory (Golden, Colo.) utilizing the same method as for corn stover.Sugar beet pulp, generated via pectinase treatment, was supplied byAtlantic Biomass, Inc. of Frederick, Md. Using a Mettler Toledo Moistureanalyzer, the dry solids were 24% in the wet corn stover, 26% in switchgrass and 3.5% in sugar beet pulp. A 100 g wet sample of corn stover orswitchgrass was resuspended in deionized water to a final volume of 420ml and the pH adjusted to 4.8 using 10 N NaOH. For beet pulp, 8.8 gramsdry solids were brought to 350 ml with deionized water and pH adjustedto 4.8 with 10N NaOH. For all feed stocks, Accellerase 1000™ (Genencor)(a cellulase enzyme complex) was used at a ratio of 0.25 ml enzyme pergram dry biomass. Samples were incubated with agitation (110 rpm) at 50°C. for 72 hours. The pH of this material was then adjusted to 7.5 withNaOH (negligible volume change), filter sterilized through a 0.22 umfilter and used in growth experiments outlined below. A sample wasreserved for determination of glucose concentration using ahexokinase-based kit from Sigma, as described below. The same set ofcellulosic feedstocks were also prepared using Celluclast™ (Novozymes)(a cellulase) as described in the previous example.

Determination of Glucose Concentrations in Various Cellulosic FeedstocksTreated with Accellerase 1000:

Glucose concentrations were determined using Sigma Glucose Assay Reagent#G3293. Samples, treated as outlined above, were diluted 400 fold and 40ul was added to the reaction. The cellulosic preparations from cornstover, switch grass and beet pulp were determined to containapproximately 23.6, 17.1 and 13.7 g/L glucose, respectively.

Determination of Growth on Cellulosic Material:

After enzymatic depolymerization of cellulosic sources to glucose,xylose, and other monosaccharides, the materials prepared above wereevaluated as feedstocks for the growth of the 24 algal strains listed inthe Table 14 and S. cerevisiae in Proteose or YPD medias respectively.The medias were designed to contain a consistent concentration ofglucose while varying the amount of cellulosic material derived fromcorn stover, switchgrass or beet pulp. A first set of Proteose and YPDmedia contained 23.6 g/L pure glucose, while a second set of mediacontained depolymerized corn stover, switchgrass and beet pulp, each ofwhich contained 23.6 g/L glucose. The switchgrass and beet pulp mediaswere supplemented with 6.5 and 9.9 g/L pure glucose to normalize glucosein all cellulosic medias at 23.6 g/L. One ml of the appropriate mediawas added to wells of a 24 well plate. S. cerevisiae grownheterotrophically at 28° C. in YPD served as inoculum (20 ul) for theyeast wells. Twenty microliters of inoculum for the 24 algal strains wasfurnished by alga cells grown mixotrophically in Proteose mediacontaining 20 g/L glucose. all cells were incubated two days in the darkon the varying concentrations of cellulosic feedstocks at 28° C. withshaking (300 rpm). Growth was assessed by measurement of absorbance at750 nm in a UV spectrophotometer. Surprisingly, all algae strains grewon the corn stover, switchgrass and beet pulp material prepared withAccellerase 1000™ or Celluclast™, including media conditions in which100% of fermentable sugar was cellulosic-derived. Under no combinationof cellulosic feedstock and depolymerization enzyme did S. cerevisiaeoutperform growth on an equivalent amount of pure glucose, indicatingthat inhibitors to yeast growth in the cellulosic material made a majorimpact on the productivity of the fermentation. Combinations of algaestrains, depolymerization enzymes and feedstocks with 100%cellulosic-derived monosaccharides that outperformed 100% pure glucoseare shown in Table 16.

TABLE 16 Combinations of algae, enzymes, and feedstocks. Feedstock andSource/ Depolymerization Enzyme Genus/Species Designation cornstover/celluclast ™ Bracteococcus minor UTEX 66 beet pulp/accellerase ™Chlorella ellipsoidea SAG 2141 switchgrass/accellerase ™ Chlorellakessleri UTEX 252 beet pulp/accellerase ™ Chlorella kessleri UTEX 397switchgrass/accellerase ™ Chlorella luteoviridis SAG 2133 beetpulp/accellerase ™ Chlorella luteoviridis SAG 2133switchgrass/accellerase ™ Chlorella luteoviridis UTEX 22 beetpulp/accellerase ™ Chlorella luteoviridis UTEX 22 cornstover/accellerase ™ Chlorella luteoviridis UTEX 22 beetpulp/accellerase ™ Chlorella protothecoides UTEX 250 beetpulp/accellerase ™ Chlorella sp. SAG 241.80 beet pulp/accellerase ™Parachlorella kessleri SAG 12.80 switchgrass/accellerase ™ Protothecamoriformis UTEX 1441 beet pulp/accellerase ™ Prototheca moriformis UTEX1441 corn stover/accellerase ™ Prototheca moriformis UTEX 1441 cornstover/celluclast ™ Prototheca moriformis UTEX 1441 cornstover/accellerase ™ Prototheca moriformis UTEX 1434 beetpulp/accellerase ™ Pseudochlorella aquatica SAG 2149

Example 26 Carbon Utilization Screens

Strains and Culture Conditions:

Seed cultures of the various strains of microalgae identified below werestarted as 1 ml liquid cultures in 24 well plates and were grownautotrophically for 48 hours in light, agitating at ˜350 rpm. Plateswere setup with 1.5% agarose-based solid Proteose media containing 1% ofglucose, glycerol, xylose, sucrose, fructose, arabinose, mannose,galactose, or acetate as the sole fixed carbon source. For each strain,5 μl of culture from the autotrophic 24 well plate was spotted onto thesolid media. Plates were incubated for 7 days in the dark at 28° C. andexamined for growth compared to a control plate containing no additionalfixed carbon. Growth was observed for each of the species tested witheach respective feedstock, as shown in Table 17 below. Growth of thesestrains on Proteose media in the dark in the absence of additional fixedcarbon either did not occur or was extremely minimal.

TABLE 17 Algal species grown on various fixed-carbon feedstocks. FixedCarbon Source Genus/Species Source/Designation Glucose Chlorellaprotothecoides UTEX 250 Glucose Chlorella kessleri UTEX 397 GlucoseChlorella sorokiniana UTEX 2805 Glucose Parachlorella kessleri SAG 12.80Glucose Pseudochlorella aquatica SAG 2149 Glucose Chlorella reisigliiCCAP 11/8 Glucose Bracteococcus medionucleatus UTEX 1244 GlucosePrototheca stagnora UTEX 1442 Glucose Prototheca moriformis UTEX 1434Glucose Prototheca moriformis UTEX 1435 Glucose Scenedesmus rubescensCCAP 232/1 Glycerol Parachlorella kessleri SAG 12.80 Glycerol Chlorellaprotothecoides CCAP 211/8d Glycerol Bracteococcus medionucleatus UTEX1244 Glycerol Prototheca moriformis UTEX 288 Glycerol Protothecamoriformis UTEX 1435 Glycerol Chlorella minutissima UTEX 2341 GlycerolChlorella sp. CCAP 211/61 Glycerol Chlorella sorokiniana UTEX 1663Xylose Chlorella luteoviridis SAG 2133 Xylose Chlorella ellipsoidea SAG2141 Xylose Pseudochlorella aquatica SAG 2149 Xylose Chlorella sp. CCAP211/75 Xylose Prototheca moriformis UTEX 1441 Xylose Protothecamoriformis UTEX 1435 Sucrose Chlorella saccharophila UTEX 2469 SucroseChlorella luteoviridis UTEX 22 Sucrose Chlorella sp. UTEX EE102 SucroseChlorella luteoviridis SAG 2198 Sucrose Bracteococcus medionucleatusUTEX 1244 Sucrose Chlorella minutissima CCALA 20024 Fructose Chlorellakessleri UTEX 398 Fructose Chlorella trebouxiodes SAG 3.95 FructoseParachlorella kessleri SAG 27.87 Fructose Chlorella luteoviridis SAG2214 Fructose Chlorella protothecoides UTEX 31 Fructose Chlorellaprotothecoides UTEX 250 Fructose Chlorella reisiglii CCAP 11/8 FructoseChlorella protothecoides CCAP 211/8d Fructose Prototheca moriformis UTEX1435 Fructose Scenedesmus rubescens CCAP 232/1 Arabinose Chlorella sp.CCAP 211/75 Mannose Chlorella kessleri UTEX 263 Mannose Chlorellasaccharophila UTEX 2911 Mannose Parachlorella kessleri SAG 12.80 MannoseChlorella sp. SAG 241.80 Mannose Chlorella angustoellipsoidea SAG 265Mannose Chlorella ellipsoidea SAG 2141 Mannose Chlorella protothecoidesUTEX 250 Mannose Chlorella emersonii CCAP 211/15 Mannose Bracteococcusminor UTEX 66 Mannose Prototheca stagnora UTEX 1442 Mannose Protothecamoriformis UTEX 1439 Mannose Chlorella cf. minutissima CCALA 20024Mannose Scenedesmus rubescens CCAP 232/1 Galactose Bracteococcus minorUTEX 66 Galactose Parachlorella kessleri SAG 14.82 GalactoseParachlorella beijerinckii SAG 2046 Galactose Chlorella protothecoidesUTEX 25 Galactose Chlorella sorokiniana UTEX 1602 GalactoseParachlorella kessleri SAG 12.80 Galactose Pseudochlorella aquatica SAG2149 Galactose Chlorella luteoviridis SAG 2214 Galactose Chlorellaellipsoidea CCAP 211/42 Galactose Chlorella ellipsoidea CCAP 211/50Galactose Chlorella protothecoides UTEX 250 Galactose Chlorellaprotothecoides UTEX 264 Galactose Bracteococcus medionucleatus UTEX 1244Galactose Prototheca moriformis UTEX 1439 Galactose Protothecamoriformis UTEX 1441 Galactose Chlorella kessleri CCALA 252 AcetateChlorella sorokiniana UTEX 1230 Acetate Chlorella sorokiniana UTEX 1810Acetate Chlorella luteoviridis UTEX 22 Acetate Parachlorella kessleriSAG 12.80 Acetate Parachlorella kessleri SAG 27.87 Acetate Chlorella sp.SAG 241.80 Acetate Chlorella luteoviridis SAG 2214 Acetate Chlorellaprotothecoides UTEX 31 Acetate Chlorella protothecoides UTEX 411 AcetateChlorella ellipsoidea CCAP 211/42 Acetate Chlorella ovalis CCAP 211/21AAcetate Chlorella protothecoides CCAP 211/8d Acetate Prototheca stagnoraUTEX 1442 Acetate Chlorella protothecoides UTEX 250 Acetate Chlorellasorokiniana CCALA 260 Acetate Chlorella vulgaris CCAP 211/79 AcetateParachlorella kessleri SAG 14.82

Example 27 Production of Renewable Diesel

Cell Production:

An F-Tank batch of Chlorella protothecoides (UTEX 250) (about 1,200gallons) was used to generate biomass for extraction processes. Thebatch (#ZA07126) was allowed to run for 100 hours, while controlling theglucose levels at 16 g/L, after which time the corn syrup feed wasterminated. Residual glucose levels dropped to <0 g/L two hours later.This resulted in a final age of 102 hours. The final broth volume was1,120 gallons. Both in-process contamination checks and a thoroughanalysis of a final broth sample failed to show any signs ofcontamination. The fermentation broth was centrifuged and drum dried.Drum dried cells were resuspended in hexane and homogenized atapproximately 1000 bar. Hexane extraction was then performed usingstandard methods, and the resulting algal triglyceride oil wasdetermined to be free of residual hexane.

Production of Renewable Diesel:

The algal triglyceride oil had a lipid profile of approximately 3%C18:0, 71% C18:1, 15% C18:2, 1% C18:3, 8% C16:0, and 2% othercomponents. The oil was first subjected to hydrocracking, resulting inan approximate 20% yield loss to water and gases. Hydroisomerization wasthen performed, with an approximate 10% loss in yield to gases. A firstdistillation was then performed to remove the naptha fraction, leavingthe desired product. Approximately 20% of the material was lost tonaptha in this first distillation. A second distillation was thenperformed at a temperature sufficient to remove fractions necessary tomeet the ASTM D975 specification but leave a bottom fraction that didnot meet the 90% point for a D975 distillation. Approximately 30% of thematerial was left in the bottom fraction in the second distillation. Theresulting material was then tested for all ASTM D975 specifications.

FIGS. 29 and 30 illustrate a gas chromatograph and a boiling pointdistribution plot, respectively, of the final renewable diesel productproduced by the method of the invention. Table 18 shows the boilingpoint distribution of the resulting renewable diesel product, and Table19 shows the results of an analysis of the final product for compliancewith the ASTM D975 specifications.

TABLE 18 Boiling point distribution of renewable diesel product.Recovered BP mass % ° C. IBP 150.4  1.0 163.6  2.0 173.4  3.0 175.2  4.0188.0  5.0 194.8  6.0 196.6  7.0 197.8  8.0 207.4  9.0 210.0 10.0 214.411.0 216.6 12.0 217.6 13.0 221.4 14.0 227.6 15.0 229.8 16.0 233.2 17.0235.8 18.0 236.8 19.0 240.2 20.0 245.6 21.0 248.0 22.0 250.2 23.0 253.624.0 255.2 25.0 256.8 26.0 261.8 27.0 264.6 28.0 266.2 29.0 268.2 30.0271.0 31.0 272.4 32.0 273.4 33.0 276.2 34.0 280.0 35.0 282.4 36.0 285.237.0 287.8 38.0 289.6 39.0 291.8 40.0 294.2 41.0 295.8 42.0 296.8 43.0297.6 44.0 298.4 45.0 299.2 46.0 299.8 47.0 300.6 48.0 301.2 49.0 302.050.0 302.6 51.0 303.2 52.0 303.8 53.0 304.4 54.0 304.8 55.0 305.2 56.0305.6 57.0 306.0 58.0 306.4 59.0 306.6 60.0 307.0 61.0 307.4 62.0 307.663.0 308.0 64.0 308.4 65.0 308.8 66.0 309.2 67.0 309.6 68.0 310.2 69.0310.6 70.0 311.2 71.0 311.6 72.0 312.2 73.0 313.0 74.0 313.6 75.0 314.276.0 314.8 77.0 315.4 78.0 315.8 79.0 316.4 80.0 317.0 81.0 317.6 82.0318.4 83.0 319.0 84.0 319.6 85.0 320.2 86.0 320.8 87.0 321.2 88.0 321.889.0 322.2 90.0 322.4 91.0 322.8 92.0 323.2 93.0 324.4 94.0 326.8 95.0329.4 96.0 333.6 97.0 339.4 98.0 346.2 99.0 362.8 FBP 401.4

TABLE 19 Analytical report for renewable diesel product using D975specifications. Method Number Test Description Results Units D93A FlashPoint (PMCC) 70 ° C. D2709 Water and Sediment 0 Vol % D86 Distillation90% (Recovered) 301.0/573.9 ° C./° F. D445 Kinematic Viscosity @ 40.0°C. (104.0° F.) 2.868 mm2/sec D482 Ash <0.001 Wt % D5453 Sulfur 2.4 ppmD130 Copper Corrosion 3 hours @ 50° C. 1b D613 Cetane Number *** >65D976 Calculated Cetane Index 71.2 D2500 Cloud Point −3 ° C. D524Ramsbottom 10% Carbon Residue 0.02 Wt % D97 Pour Point −3 ° C. D2274Total Insolubles (Oxidation Stability) 40 Hour Test *** 4.0 mg/100 mLD4052 Density @ 15.0° C. (59.0° F.) 793.8 kg/m³ D4176-1 Appearance byVisual Inspection (Lab) Clear and Bright- Pass Visual D4176-1 Appearanceby Visual Inspection (Lab) Free Water- Pass Visual D4176-1 Appearance byVisual Inspection (Lab) Particulates- Pass Visual D1500 ASTM Color L 0.5D664 Acid Number <0.10 mg KOH/g D6079 Lubricity (Wear Scar) 405 μm

What is claimed is:
 1. A method of producing lipid, the method comprising: preparing a culture medium comprising depolymerized cellulosic material as a primary carbon source, the depolymerized cellulosic material comprising glucose and xylose, wherein the cellulosic material is sugarcane bagasse, rice hulls, corn fiber, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, paper fractions of municipal solid waste, grass clippings, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum; placing a recombinant microalgae cell of the species Prototheca moriformis comprising an exogenous gene encoding a fatty acyl-ACP thioesterase in the culture medium; culturing the recombinant microalgae cell under heterotrophic growth and limited nitrogen conditions, and until the microalgae cell accumulates at least 60% of its dry cell weight as lipid; and obtaining the lipid from the recombinant microalgae cell.
 2. The method of claim 1, wherein the culture is axenic.
 3. The method of claim 1, wherein the lipid is characterized by a lipid profile that comprises at least 3% C18:0 and at least 8% C16:0.
 4. The method of claim 1, wherein the source of depolymerized cellulosic material is sugarcane bagasse.
 5. The method of claim 1, wherein the source of depolymerized cellulosic material is corn fiber.
 6. The method of claim 1, wherein the source of depolymerized cellulosic material is miscanthus.
 7. The method of claim 1, wherein the culture medium further comprises at least one additional carbon source selected from the group consisting of sucrose, glycerol, fructose, arabinose, mannose, galactose and acetate. 